FORMULATION AND STABILITY TESTING OF EYE DROP ...

FORMULATION AND STABILITY TESTING OF EYE DROP PREPARATIONS

CONTAINING PHENYLEPHRINE HYDROCHLORIDE

CHINEDUM OLUCHUKWU OKAFOR

ii

FORMULATION AND STABILITY TESTING OF EYE DROP PREPARATIONS

CONTAINING PHENYLEPHRINE HYDROCHLORIDE

CHINEDUM OLUCHUKWU OKAFOR

Submitted in fulfillment of the requirements for the degree of MAGISTER

SCIENTIAE in the FACULTY OF HEALTH SCIENCES at the NELSON MANDELA

METROPOLITAN UNIVERSITY

DECEMBER 2012

SUPERVISOR: Mrs. M. Keele

CO-SUPERVISORS: Dr M. Worthington, Prof. G. Kilian

iii

DECLARATION

I, Chinedum Oluchukwu Okafor, 205010351, hereby declare that the dissertation for

Magister Scientiae is my own work and that it has not previously been submitted for

assessment or completion of any postgraduate qualifcaton to another University or

for another qualification.

Chinedum Oluchukwu Okafor

iv

ACKNOWLEDGEMENTS

I wish to thank the following people and institutions for their assistance during the

compiling of this dissertation:

My exceptional families Okafor and Makamure for their steadfast support and

love;

Dr. M Worthington, as without sponsorship and guidance there could be no

research;

My supervisors, Mrs. M. Keele, and Prof. G. Kilian for guidance and support;

Prof. Milne, for his unwavering support, no words in the dictionary can

describe his help;

Michael (Aspen), Jean, Charne and Arista (NMMU) for her input, support and

exceptional skills at sourcing materials for me;

Aspen Pharmacare, for financial assistance and the use of equipment and

materials needed to perform my experiments;

All my friends all around the world, every moment with you was a blessing.

The Pharmacy, Biochemistry and Microbiology and Chemistry Departments of

the Nelson Mandela Metropolitan University for the use of laboratory facilities

and technical assistance;

Above all, God Almighty, only through His Grace can I achieve all things.

v

SUMMARY

Phenylephrine hydrochloride is a potent adrenergic agent and β-receptor

sympathomimetic drug, used in its optically active form (Pandey et al., 2003; Pandey

et al., 2006). As an α1-adrenergic receptor agonist, phenylephrine hydrochloride is

used ocularly as a decongestant for uveitis and as an agent to dilate the pupil (Lang,

1995). High intraocular doses have been reported to cause tachycardia,

hypertension, and headache. These side effects are caused by large amounts of the

drop draining into the nasal cavity. Eye drops that contain phenylephrine

hydrochloride have proven to have low intra-ocular bioavailability because of a short

contact time with the eyes which reduces the amount of drug reaching the site of

action. Formulations of phenylephrine hydrochloride eye drops have varying shelf-

lives of approximately two to four years. The aim of this study was to formulate and

manufacture an eye drop product containing phenylephrine hydrochloride. Important

characteristics that were targeted were increased ocular absorption by increasing the

viscosity of the product and reduced degradation of phenylephrine hydrochloride.

A variety of phenylephrine hydrochloride formulations were manufactured on a

laboratory scale using hydroxypropyl methylcellulose (HPMC), glycerol, and sodium

carboxy methylcellulose as viscosity modifying agents (VMA). The concentration of

phenylephrine hydrochloride was ten percent. Ten millimeters of each formulation

was made in triplicate. The quantity in each was evaluated using a previously

validated high performance (pressure) liquid chromatography method.

Physicochemical properties including pH and colour were also evaluated. Stability

was assessed using real time and accelerated stability conditions in accordance with

the International Conference on Harmonization (ICH) guidelines.

Formulations containing hydroxypropyl methylcellulose (HPMC) as the viscosity

modifying agents proved to be stable under all storage conditions when compared

with formulations containing other viscosity modifying agents (VMA). However,

sodium citrate dihydrate; sodium metabisulphite and EDTA also stabilized the

formulations to a certain extent.

vi

Changes in the appearance and colour of products containing glycerol under

accelerated storage conditions were observed. The sodium carboxy methylcellulose

(SCMC) containing formulation was found to be physically and chemically stable in

two conditions, namely 30 °C/65%RH and 25 °C/60%RH. The formulations

containing hydroxypropyl methylcellulose along with an antioxidant showed to be

most stable as it remained aesthetically pleasing did not change colour and did not

have a reduction in phenylephrine hydrochloride concentrations. This meant that

phenylephrine hydrochloride did not degrade while the viscosity modifying agents

remained stable.

Rheological tests showed differences in the viscosities of the formulations as

glycerol had increased in viscosity over time while HMPC and SCMC displayed

relative similarities. The formulations were compared to a marketed eye drop

containing polyvinyl alcohol as a VMA. After rheological analysis the formulation

containing HPMC displayed better viscosity than the product with polyvinyl alcohol.

The preservatives in the formulations were active against the microbial organisms

use to challenged them.

Key words: Phenylephrine hydrochloride, glycerol, hydroxypropyl methylcellulose,

preservatives, storage conditions, viscosity.

vii

TABLE OF CONTENTS

DECLARATION ...................................................................................................................... iii

ACKNOWLEDGEMENTS ....................................................................................................... iv

SUMMARY ............................................................................................................................... v

TABLE OF CONTENTS ......................................................................................................... vii

LIST OF ABBREVIATIONS ..................................................................................................... x

LIST OF FIGURES ............................................................................................................... xiv

LIST OF TABLES ............................................................................................................... xxviii

1. INTRODUCTION ............................................................................................................. 1

1.1 Background and motivation ...................................................................................... 1

1.2 Aim and objectives ................................................................................................... 2

1.3 Plan of work .............................................................................................................. 3

2. LITERATURE REVIEW ................................................................................................... 4

2.1 Anatomy and physiology of the eye ......................................................................... 4

2.2 Pathophysiology of the eye ......................................................................................... 10

2.3 Phenylephrine hydrochloride and its ocular uses ........................................................ 14

2.3.1 Phenylephrine hydrochloride .................................................................................... 14

2.3.2 Pharmacological actions and uses ........................................................................... 15

2.3.3 Mechanism of action ................................................................................................ 15

2.3.4 Pharmacokinetics ..................................................................................................... 16

2.3.5 Adverse effects ......................................................................................................... 16

2.3.6 Drug interactions ...................................................................................................... 17

2.3.7 Bioavailability ............................................................................................................ 17

2.3.7.1 Reasons for poor ocular bioavailability .................................................................. 18

2.3.7.2 Strategies for improving drug availability in ocular adminstration ......................... 19

2.3.7.2.1 Increasing ocular residence time ........................................................................ 19

2.3.7.2.2 Increasing ocular absorption .............................................................................. 19

2.3.7.2.3 Altering drug structure ........................................................................................ 20

2.3.8 Polymorphism and pseudomorphism of phenylephrine hydrochloride ..................... 21

2.4 Ophthalmic formulations .............................................................................................. 21

2.4.1 Eye drops as an ophthalmic dosage form ................................................................ 22

2.4.2 Eye drop formulation characteristics ........................................................................ 24

2.4.2.1 Clarity .................................................................................................................... 24

2.4.2.2 Stability, pH and buffer systems ............................................................................ 25

2.4.2.3 Tonicity .................................................................................................................. 26

viii

2.4.2.4 Viscosity ................................................................................................................ 27

2.4.2.5 Additives ................................................................................................................ 30

2.5 Sterilization .................................................................................................................. 34

2.5.1 Steam under pressure as a method of sterilization .................................................. 35

2.5.2 Filtration as a method of sterilization ........................................................................ 35

2.5.3 Laminar-flow principles ............................................................................................. 35

2.5.4 Preservatives used in eye drop formulations ........................................................... 36

2.5.4.1 Quaternary ammonium compounds ...................................................................... 38

2.5.4.2 Parahydroxybenzoic acid esters ........................................................................... 40

2.6 Efficacy of antimicrobial preservation .......................................................................... 42

2.7 Packaging .................................................................................................................... 43

2.9 Formulation development ............................................................................................ 44

2.9.1 Validation of HPLC analytical methods .................................................................... 46

2.9.1.1 Stability indicating HPLC analysis ......................................................................... 47

2.9.1.2 Choice of analytical column and conditions ........................................................... 48

2.9.1.3 Steps for HPLC method validation ........................................................................ 50

2.9.1.4 Linearity ................................................................................................................. 50

2.9.1.5 Accuracy and precision ......................................................................................... 51

2.9.1.6 Limit of detection and limit of quantification ........................................................... 51

2.9.1.7 Range .................................................................................................................... 51

2.9.1.8 Specificity .............................................................................................................. 51

2.9.2 Active-excipient compatibility studies ....................................................................... 53

2.10 Determining formulation stability study ...................................................................... 54

3. METHODOLOGY .......................................................................................................... 57

3.1 HPLC method validation .............................................................................................. 57

3.1.1 Equipment ................................................................................................................ 57

3.1.2 Materials and reagents ............................................................................................. 57

3.1.3 Mobile phase preparation and standard curve construction ..................................... 57

3.1.4 Chromatographic conditions ..................................................................................... 58

3.1.5 Linearity .................................................................................................................... 58

3.1.6 Accuracy and precision ............................................................................................ 59

3.1.7 Limit of detection and limit of quantification .............................................................. 59

3.1.8 Range and system suitability .................................................................................... 60

3.1.9 Specificity ................................................................................................................. 60

3.2 Determination of active–excipient compatibility .......................................................... 62

3.3 Manufacture of products .............................................................................................. 62

ix

3.3.1 Materials ............................................................................................................... 63

3.3.2 Product manufacture ................................................................................................ 63

3.3.2.1 Sterilization for heat sensitive API and exipients ................................................... 64

3.3.3 Manufacturing methods for products I–V ................................................................. 64

3.4 Stability Tests .............................................................................................................. 69

3.5 Qualitative and quantitative analysis of the formulations ............................................ 70

3.5.1 Appearance and pH ................................................................................................. 70

3.5.2 Phenyleprine hydrochloride concentration ............................................................... 70

3.6 Test for preservative efficacy ...................................................................................... 70

3.6.1 Procedure for standard plate count .......................................................................... 71

3.6.2 Procedure for plating the bacteria and fungi ............................................................ 71

3.6.3 Standardization of cultures using turbidimetry method ............................................ 72

3.6.4 Preservative efficacy ................................................................................................ 73

3.7 Determination of viscosity ........................................................................................... 73

3.8 Statistical analysis ....................................................................................................... 74

4. RESULTS AND DISCUSSION ...................................................................................... 75

4.1 Validation of the stability indicating assay ................................................................... 75

4.1.1 Linearity .............................................................................................................. 75

4.1.2 Accuracy .......................................................................................................... 76

4.1.3 Precision ......................................................................................................... 76

4.1.4 Limit of detection (LOD) and quantification (LOQ) .......................................... 77

4.1.5 Specificity and system suitability ..................................................................... 77

4.2 Active and excipient study ......................................................................................... 113

4.3 Stability study ............................................................................................................ 123

4.4 Determination of yield point and viscosity of products .............................................. 137

4.5 Effectiveness of the ophthalmic solution preservatives ............................................. 144

5. CONCLUSION AND RECOMMENDATIONS .............................................................. 147

REFERENCES ................................................................................................................... 151

APPENDIX A ...................................................................................................................... 176

CONCEPT ARTICLE ....................................................................................................... 176

APPENDIX B ...................................................................................................................... 192

LIST OF EQUIPMENT .................................................................................................... 192

APPENDIX C ...................................................................................................................... 193

LIST OF SOLUTIONS ..................................................................................................... 193

x

LIST OF ABBREVIATIONS

API Active Pharmaceutical Ingredient

AUC Area under Curve

Å angstrom

ANOVA Analysis of variance

atm atmospheric pressure

BP British Pharmacopeia

CPR Cardio Pulmonary Resuscitation

COMT catechol–O–methyltransferases

R2 Correlation coefficient

cAMP cyclic Adenosine Monophosphate

CYP Cytochrome P 450

Da Dalton

EDTA Ethylenediaminetetraacetic acid

ET Eustachian tube

FPLC Fast Protein Liquid Chromatography

FDA Food and Drug Administration

xi

> Greater than

HEPA High Efficiency Particulate Air

HPLC High Performance (pressure) Liquid Chromatography

HIV Human Immunodeficiency Virus

HPMC Hydroxypropyl methylcellulose

ICH International Conference on Harmonization

kg kilogram

< Less than

log logarithmic

m/v Mass per volume

MCC Medicines Control Council

MAO Monoamine oxidases

MIC Minimum Inhibitory Concentration

µg microgram

µL microliter

µm micrometer

mg milligram

xii

mm millimeter

min minute

M Molar concentration (moles of solute per liter of solution)

N Normal concentration (gram-equivalents of solute per liter of

solution)

OTC Over–The–Counter

Pa·s Pascals per second

PAC Perennial Allergic Conjunctivitis

% Percentage

psi Pounds per square inch

RH Relative humidity

RSD Relative Standard Deviation

~ roughly similar

SAC Seasonal Allergic Conjunctivitis

s seconds

SA South Africa

SD Standard Deviation

xiii

Tf Tailing factor

T Temperature

TPN Total Parenteral Nutrition

UV Ultraviolet

USP United States Pharmacopeia

UK United Kingdom

VMA Viscosity Modifying Agent

λ Wavelength

xiv

LIST OF FIGURES

Figure 1: Anatomy of the eye (Del Amo & Urtti, 2008). ............................................. 4

Figure 2: Structure of Phenylephrine, its base and salts (Trommer et al., 2010). .... 15

Figure 3: Diagram of Ocular Absorption (Nanjawade et al., 2007). ......................... 17

Figure 4: Diagram of a typical HPLC-UV absorbance peak and plots of noise (or

threshold) and purity angles (Krull & Swartz, 2001). ................................................ 52

Figure 5: Laboratory scale 1000 ml manufacturing process of product I ................. 65

Figure 6: Laboratory scale 1000 ml manufacturing process of product II ................ 66

Figure 7: Laboratory scale 1000 ml manufacturing process of product III ............... 67

Figure 8: Laboratory scale 1000 ml manufacturing process of product IV ............... 68

Figure 9: Laboratory scale 1000 ml manufacturing process of product V ................ 69

Figure 10: Graph showing a mean peak area versus concentration of replicate

samples of phenylephrine hydrochloride standards. Linear regression equation: y =

8541.1x + 438.55, R2 = 0.9999. ............................................................................... 75

Figure 11: HPLC Chromatogram for mobile phase alone. ....................................... 78

Figure 12: HPLC Chromatogram for phenylephrine hydrochloride dissolved in

mobile phase with a retention time of 7.80 minutes. ................................................ 79

Figure 13: Peak purity profile calculated using PDA data (from 190–800 nm) for

phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and

purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999.

................................................................................................................................. 79

Figure 14: HPLC Chromatogram for product I dissolved in mobile phase with a

retention time of 7.87 minutes. ................................................................................. 79


Product I prepared in mobile phase. Peak shown in pink and purity curve in black.

Peak purity index = 1.00000; Single point threshold = 0.999054 ............................. 80

Figure 16: HPLC Chromatogram for product II dissolved in mobile phase with a



Product II prepared in mobile phase. Peak shown in pink and purity curve in black.

Peak purity index = 1.00000; Single point threshold = 0.996482. ............................ 80

xv

Figure 18: HPLC Chromatogram for product III dissolved in mobile phase with a



Product III prepared in mobile phase. Peak shown in pink and purity curve in black.


Figure 20: HPLC Chromatogram for product IV dissolved in mobile phase with a



Product IV prepared in mobile phase. Peak shown in pink and purity curve in black.


Figure 22: HPLC Chromatogram for product V dissolved in mobile phase with a



product V prepared in mobile phase. Peak shown in pink and purity curve in black.


Figure 24: HPLC Chromatogram for phenylephrine hydrochloride stressed under UV

light dissolved in mobile phase with a retention time of 7.81 minutes. ..................... 83


phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and


................................................................................................................................. 83

Figure 26: HPLC Chromatogram for product I stressed under UV light dissolved in



product I prepared in mobile phase. Peak shown in pink and purity curve in black.


Figure 28: HPLC Chromatogram for product II stressed under UV light dissolved in



product II in mobile phase. Peak shown in pink and purity curve in black. Peak purity

index = 1.00000; Single point threshold = 0.999116. ............................................... 85

Figure 30: HPLC Chromatogram for product III stressed under UV light dissolved in


xvi


product III in mobile phase. Peak shown in pink and purity curve in black. Peak purity

index = 0.999999; Single point threshold = 0.997116. ............................................. 85

Figure 32: HPLC Chromatogram for product IV stressed under UV light dissolved in



product IV in mobile phase. Peak shown in pink and purity curve in black. Peak

purity index = 1.000000; Single point threshold = 0.997916. .................................... 86

Figure 34: HPLC chromatogram for product V stressed under UV light dissolved in



product V in mobile phase. Peak shown in pink and purity curve in black. Peak purity

index = 1.000000; Single point threshold = 0.997475. ............................................. 87

Figure 36: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M

HCl dissolved in mobile phase with a retention time of 7.86 minutes. ...................... 88


phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in

black. Peak purity index = 1.000000; Single point threshold = 0.999574. ................ 88

Figure 38: HPLC chromatogram of Product I stressed with 0.2 M HCl dissolved in



product I stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity

curve in black. Peak purity index = 0.999999; Single point threshold = 0.997116. ... 89

Figure 40: HPLC chromatogram for product II stressed with 0.2 M HCl dissolved in



product II stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity


Figure 42: HPLC chromatogram for product III stressed with 0.2 M HCl dissolved in



product III stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity

curve in black. Peak purity index =0.999999; Single point threshold = 0.995179. .... 90

xvii

Figure 44: HPLC chromatogram for product IV stressed with 0.2 M HCl dissolved in



product IV stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity


Figure 46: HPLC chromatogram of product V stressed with 0.2 M HCl dissolved in



product V stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity


Figure 48: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M

NaOH dissolved in mobile phase with a retention time of 7.82 minutes ................... 92


phenylephrine hydrochloride stressed with 0.2 M NaOH in mobile phase. Peak

shown in pink and purity curve in black. Peak purity index =0.999999; Single point

threshold = 0.996847. .............................................................................................. 92

Figure 50: HPLC chromatogram of product I stressed with 0.2 M NaOH dissolved in



product I stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity


Figure 52: HPLC chromatogram of product II stressed with 0.2 M NaOH dissolved in

mobile phase with a retention time of 7.9 minutes. .................................................. 93


product II stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity


Figure 54: HPLC chromatogram for product III stressed with 0.2 M NaOH dissolved

in mobile phase with a retention time of 7.79 minutes. ............................................. 94


product III stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity


Figure 56: HPLC chromatogram of product IV stressed with 0.2 M NaOH dissolved


xviii


product IV stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and

purity curve in black. Peak purity index = 0.999999; Single point threshold =

0.996930. ................................................................................................................. 95

Figure 58: HPLC chromatogram for product V stressed with 0.2 M NaOH dissolved



product V stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity


Figure 60: HPLC chromatogram for phenylephrine hydrochloride stressed with 0.2

M H2O2 dissolved in mobile phase with a retention time of 7.82 minutes. ................ 96


phenylephrine hydrochloride stressed with 0.2 H2O2 in mobile phase. Peak shown in

pink and purity curve in black. Peak purity index = 1.000000; Single point threshold =

0.999987. ................................................................................................................. 97

Figure 62: HPLC chromatogram for product I stressed with 0.2 M H2O2 dissolved in



product I stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity


Figure 64: HPLC chromatogram for product II stressed with 0.2 M H2O2 dissolved in

mobile phase with a retention time of 7.9 minutes. .................................................. 98


product II stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity


Figure 66: HPLC chromatogram of product III stressed with 0.2 M H2O2 dissolved in

mobile phase with a retention time 7.94 minutes. .................................................... 98


product III stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity


Figure 68: HPLC chromatogram for product IV with 0.2 M H2O2 dissolved in mobile

phase with a retention time of 7.81 minutes. ............................................................ 99

xix


product IV stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity


Figure 70: HPLC chromatogram for product V stressed with 0.2 M H2O2 dissolved in

mobile phase with a retention time of 7.88 minutes. .............................................. 100


product V stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity

curve in black. Peak purity index = 0.999999; Single point threshold = 0.999536. . 100

Figure 72: HPLC chromatogram for phenylephrine hydrochloride stored at 100 °C

for 24 hours dissolved in mobile phase with a retention time of 7.8 minutes. ......... 102


phenylephrine hydrochloride stored at 100 °C for 24 hours in mobile phase. Peak

shown in pink and purity curve in black. Peak purity index = 1.000000; Single point

threshold = 0.999999. ............................................................................................ 102

Figure 74: HPLC chromatogram for phenylephrine hydrochloride stored at 65 °C for

1 month dissolved in mobile phase with a retention time of 7.83 minutes. ............. 103


phenylephrine hydrochloride stored at 65 °C for 1 month in mobile phase. Peak


threshold = 0.999989. ............................................................................................ 103

Figure 76: HPLC chromatogram for product I stored at 65 °C for 1 month dissolved

in mobile phase with a retention time of 7.92 minutes. ........................................... 103


product I stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity


Figure 78: HPLC chromatogram for product II stored at 65 °C for 1 month dissolved



product II stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity


Figure 80: HPLC chromatogram for Product III stored at 65 °C for 1 month dissolved



product III stored at 65 °C for 1 month in mobile phase. Peak shown in pink and

xx


0.999720. ............................................................................................................... 105

Figure 82: HPLC chromatogram for Product IV stored at 65 °C for 1 month dissolved



product IV stored at 65 °C for 1 month in mobile phase. Peak shown in pink and

purity curve in black. Peak purity index =0.999999; Single point threshold =

0.999803. ............................................................................................................... 106

Figure 84: HPLC chromatogram for Product V stored at 65 °C for 1 month dissolved



product V stored at 65 °C for 1 month in mobile phase. Peak shown in pink and


0.999752. ............................................................................................................... 106

Figure 86: HPLC chromatogram for phenylephrine hydrochloride stored at 40

°C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.86

minutes. .................................................................................................................. 107

Figure 87: Peak purity profile calculated using PDA date (from 190–800 nm) for

phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month in mobile phase.

Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single

point threshold = 0.999056. .................................................................................... 107

Figure 88: HPLC chromatogram for Product I stored at 40 °C/75%RH for 1 month

dissolved in mobile phase with a retention time of 7.87 minutes. ........................... 107


product I stored at 40 °C/75% RH for 1 month in mobile phase. Peak shown in pink

and purity curve in black. Peak purity index = 0.999999; Single point threshold =

0.995566. ............................................................................................................... 108

Figure 90: HPLC chromatogram for Product II stored at 40 °C/75%RH for 1 month



product II stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink


0.999579. ............................................................................................................... 108

xxi

Figure 92: HPLC chromatogram for product III stored at 40 °C/75%RH for 1 month



product III stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink


0.994609. ............................................................................................................... 109

Figure 94: HPLC chromatogram for Product IV stored at 40 °C/75%RH for 1 month



product IV stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink


0.994609. ............................................................................................................... 110

Figure 96: HPLC chromatogram for Product V stored at 40 °C/75%RH for 1 month



product V stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink


0.995332. ............................................................................................................... 110

Figure 98: HPLC chromatogram for phenylephrine hydrochloride alone dissolved in

mobile phase with retention of 7.82 minutes. ......................................................... 115


phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in

black. Peak purity index = 1.000000; Single point threshold = 0.999999. .............. 115

Figure 100: HPLC chromatogram for phenylephrine hydrochloride with sodium

citrate dihydrate (1:1) dissolved in mobile phase with a retention time of 7.92

minutes. .................................................................................................................. 115


phenylephrine hydrochloride with sodium citrate dihydrate (1:1) in mobile phase.

Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single

point threshold = 0.999896. .................................................................................... 116

Figure 102: HPLC chromatogram for phenylephrine hydrochloride with

carboxymethycellulose sodium (1:1) dissolved in mobile phase with a retention time

of 7.82 minutes. ...................................................................................................... 116

xxii


phenylephrine hydrochloride with carboxymethycellulose sodium (1:1) in mobile

phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000;

Single point threshold = 0.999999. ......................................................................... 116

Figure 104: HPLC chromatogram for phenylephrine hydrochloride with hypromellose

(1:1) dissolved in mobile phase with a retention time of 7.81 minutes. .................. 117


phenylephrine hydrochloride with hypromellose (1:1) in mobile phase. Peak shown in


0.999999. ............................................................................................................... 117

Figure 106: HPLC chromatogram for phenylephrine hydrochloride with glycerol (1:1)



phenylephrine hydrochloride with glycerol (1:1) in mobile phase. Peak shown in pink


0.999989. ............................................................................................................... 118

Figure 108: HPLC chromatogram for phenylephrine hydrochloride with

benzalkonium chloride (1:1) dissolved in mobile phase with a retention time of 7.88

minutes. .................................................................................................................. 118


phenylephrine hydrochloride with benzalkonium chloride (1:1) in mobile phase. Peak


threshold = 0.999918. ............................................................................................ 118

Figure 110: HPLC chromatogram for phenylephrine hydrochloride with EDTA (1:1)



phenylephrine hydrochloride with EDTA (1:1) in mobile phase. Peak shown in pink


0.999991. ............................................................................................................... 119

Figure 112: HPLC chromatogram for phenylephrine hydrochloride with boric acid

(1:1) dissolved in mobile phase with a retention time of 7.92 minutes. .................. 119


phenylephrine hydrochloride with boric acid (1:1) in mobile phase. Peak shown in

xxiii


0.999999. ............................................................................................................... 120

Figure 114: HPLC chromatogram for phenylephrine hydrochloride with sodium

metabisulfite (1:1) dissolved in mobile phase with a retention time of 7.82 minutes.

............................................................................................................................... 120


phenylephrine hydrochloride with sodium metabisulfite (1:1) in mobile phase. Peak


threshold = 0.999870. ............................................................................................ 120

Figure 116: HPLC chromatogram for phenylephrine hydrochloride with disodium

edetate (1:1) dissolved in mobile phase with a retention time of 7.82 minutes. ...... 121


phenylephrine hydrochloride with disodium edetate (1:1) in mobile phase. Peak


threshold = 0.998891. ............................................................................................ 121

Figure 118: HPLC chromatogram for phenylephrine hydrochloride with propyl

paraben (1:1) dissolved in mobile phase with a retention time of 7.83 minutes. .... 121


phenylephrine hydrochloride with propyl paraben (1:1) in mobile phase. Peak shown

in pink and purity curve in black. Peak purity index = 1.000000; Single point

threshold = 0.999948. ............................................................................................ 122

Figure 120: HPLC chromatogram for phenylephrine hydrochloride with methyl

paraben (1:1) dissolved in mobile phase with a retention time of 7.82 minutes. .... 122


phenylephrine hydrochloride with methyl paraben (1:1) in mobile phase. Peak


threshold = 0.999982. ............................................................................................ 122

Figure 122: HPLC chromatogram for product I stored at 30 °C/65%RH for 3 months



product I stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink


0.999950. ............................................................................................................... 124

xxiv

Figure 124: HPLC chromatogram for product II stored at 30 °C/65%RH for 3 months



product II stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink


0.996473. ............................................................................................................... 124

Figure 126: HPLC chromatogram for product III stored at 30 °C/65%RH for 3

months dissolved in mobile phase with a retention time of 7.87 minutes. .............. 125


product III stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink


0.996955. ............................................................................................................... 125

Figure 128: HPLC chromatogram for product IV stored at 30 °C/65% RH for 3



product IV stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink


0.998694. ............................................................................................................... 126

Figure 130: HPLC chromatogram for product V stored at 30 °C/65%RH for 3 months



product V stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink


0.994533. ............................................................................................................... 126






0.995873. ............................................................................................................... 127





xxv


0.999950. ............................................................................................................... 128






0.998742. ............................................................................................................... 129

Figure 138: HPLC chromatogram for product IV stored at 25 °C/60%RH for 3





0.996396. ............................................................................................................... 129






0.998093. ............................................................................................................... 130



Figure 143: Peak purity profile calculated using PDA date (from 190 – 800 nm) for



0.998093. ............................................................................................................... 131



Figure 145: Peak purity profile calculated using PDA date (from 190 – 800 nm) for



0.998093. ............................................................................................................... 132



xxvi




0.998093. ............................................................................................................... 132

Figure 148: HPLC chromatogram for product IV stored at 40 °C/75%RH for 3





0.998093. ............................................................................................................... 133






0.998093. ............................................................................................................... 134

Figure 152: A graph showing standard error and phenylephrine hydrochloride left in

product I–V after 12 weeks at 30 °C/65%RH, 40 °C/75%RH, 25 °C/60%RH. ........ 134

Figure 153: Flow and viscosity curve of Prefrin® and products I–V at time zero for

storage condition 30 °C/65% RH. ........................................................................... 138

Figure 154: Flow and viscosity curve of Prefrin® and products I–V after 3 months for







storage condition 25 °C/60%RH. ............................................................................ 140



Figure 159: Graph showing viscosity of products I–V stored in a stability chamber of

40 °C/75%RH tested at time zero (T0), three months later (T1) and compared to an

original marketed product Prefrin®. ........................................................................ 141

xxvii


25 °C/60%RH tested at time zero (T0), 3 months later (T1) and compared to an



30 °C/65%RH tested at time zero (T0), 3 months later (T1) and compared to an


xxviii

LIST OF TABLES

Table 1: Conventional dosage forms and usage (Lang, 1995). ............................... 24

Table 2: Typical minimum inhibitor concentrations of benzalkonium chloride (Kibbe,

2006). ....................................................................................................................... 39

Table 3: Minimum inhibitory concentration for propylparaben in aqueous solution

(Rieger, 2006b) ........................................................................................................ 40

Table 4: Minimum inhibitory concentrations of methylparaben in aqueous solution

(Rieger, 2006a). ....................................................................................................... 41

Table 5: Formulation summary of active pharmaceutical ingredient and excipients

used in the manufacturing of products I–V ............................................................... 64

Table 6: Criteria for tested microorganisms (USP, 2004) ........................................ 73

Table 7: Accuracy data for quantification of phenylephrine hydrochloride ............... 76

Table 8: Precision data for quantification of phenylephrine hydrochloride ............... 76

Table 9: Physical appearance of phenylephrine hydrochloride and products I–V

before and after storage conditions 40 °C/75%RH for 1 month .............................. 101

Table 10: Results showing absence of impurity from a series of stressed and

unstressed samples of phenylephrine hydrochloride (API) and products. .............. 111

Table 11: Results showing phenylephrine hydrochloride left with samples stressed

and unstressed (API and Products) ....................................................................... 112

Table 12: Assay result showing phenylephrine hydrochloride and excipients in a 1:1

ratio after storage conditions 40 °C/75%RH for 1 month ........................................ 113

Table 13: Physical appearance of active–excipients samples before and after

storage conditions 40 °C/75%RH for 4 weeks ........................................................ 114

Table 14: Results of one-way ANOVA analysis for products I–V stored at 25

°C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months. ................................. 135

Table 15: Changes in pH of phenylephrine hydrochloride and products I–V before

and after storage conditions 40 °C/75%RH for 1 month. ........................................ 136

Table 16: Changes in pH for products I–V before and after varying storage

conditions for 3 months .......................................................................................... 136

Table 17: Results of one-way ANOVA analysis for similarities in pH of products II 25

°C/60%RH, 40 °C/75%RH and IV 25 °C/60%RH after 3 months. .......................... 136

xxix

Table 18: Physical appearance of products I–V before and after varying storage

conditions for 3 months. ......................................................................................... 137

Table 19: Results of one-way ANOVA analysis for viscosity of products I–V stored at

25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months. The values shown

indicate differences in p-values and significance in differences of mean was defined

as p < 0.05. ............................................................................................................ 143

Table 20: Antimicrobial preservative efficacy of the eye-drop products I–V

challenged with E. coli, S.aureus, P. aeruginosa, C.albicans. ................................ 145

1

1. INTRODUCTION

1.1 Background and motivation

Phenylephrine is a sympathomimetic amine drug that undergoes extensive first pass

metabolism resulting in a bioavailability of approximately 38% or lower (Trommer et

al., 2010). Phenylephrine hydrochloride ((R)-1-(3-hydroxyphenyl)-2-methyl-

aminoethanol hydrochloride) is an effective adrenergic agent and β–receptor

sympathomimetic drug that is chemically related to epinephrine (Ahmed and Amin,

2007) and used in its optically active form (Pandey et al., 2003; Pandey et al., 2006).

As an α1-adrenergic receptor agonist it is used primarily as a decongestant, for

uveitis and as an agent to dilate the pupil (Lang, 1995).

Instilling pupil-dilating agents like phenylephrine hydrochloride allows for maximum

dilation of the pupil during ophthalmic examinations as well as during many ocular

surgical procedures (Hanyu et al., 2007). For the period of an ophthalmoscopic

examination, a perfectly dilated pupil should be large and stable to the intensive light

stimulation. A high frequency of drug instillation is needed to produce a satisfactory

response due to the rapid clearance of phenylephrine hydrochloride from the

ophthalmic surface by the lachrimal system (Zoukhri, 2006). Thus a patient may

receive up to 30 drops of phenylephrine hydrochloride during an ophthalmic

procedure to maintain an optimal pupil size (Hanyu et al., 2007). Differences in the

range of drops received are due to patient’s interdependent variability as not all

patients are the same. The doses of phenylephrine hydrochloride administered

ocularly could precipitate unwanted side effects, posing a problem for both physician

and patient.

The conjunctival sac holds a limited capacity of fluids which poses another problem,

as most of the eye drop solution is drained into the nasal cavity thereby reducing the

portion of drug that reaches its site of action. Systemic absorption of phenylephrine

hydrochloride into the nasal mucosa may produce unwanted side effects such as

headache, hypertension and tachycardia (Bartlett & Jaanus, 2008). Moreover,

phenylephrine hydrochloride solutions may irritate the eye due to the presence of

2

preservatives such as benzalkonium chloride and methyl paraben amongst others

(Giaconi et al., 2009).

The contact time of eye drops is considered as being the most important factor in

ophthalmic drug delivery (Agarwal et al., 2002). This research study will focus on

formulating an eye drop solution which improves contact time. Various formulations

of phenylephrine hydrochloride eye drops have been made in the quest to improve

contact time. These include viscous solutions (Saettone et al., 1984), rods (Alani,

1978), gels (Durrani et al., 1996), ointments (Saettone et al., 1980; Gurjar et al.,

1998), and a polyvinyl alcohol flag (O’Donnell & Gillibrand, 1995; Maitani et al.,

1997).

Phenylephrine hydrochloride have been formulated in varying dosage forms using

potassium, sodium and lysine salts or mixed with viscosity modifiers such as HPMC

and SCMC to improve its effectiveness. Ocular penetration and retention of

phenylephrine hydrochloride demands an ophthalmic solution of acidic pH which

could precipitate the drug or increase the ocular irritation potential and viscosity

modifiers could solve both problems. Benzalkonium chloride, a cationic preservative,

used in eye drops causes eye irritation, caution is needed in its use and

concentration. Thus a non irratiting ophthalmic solution of phenylephrine

hydrochloride can be formulated by dissolving an eye-friendly water soluble salt, a

viscosity modifyier, an antioxidant in purified water and stability in mind with

benzalkonium chloride.

However, there is still scope for an eye drop that can be administered easily in less

frequent doses which produces consistent, rapid results and minimizes the risk of

adverse effects to the patient.

1.2 Aim and objectives

The primary aim of the study was to develop a pharmaceutically stable

phenylephrine hydrochloride eye drop. The following objectives were accordingly

identified:

3

Validate a high performance liquid chromatographic (HPLC) method for the

quantitative determination of phenylephrine hydrochloride in the finished

product.

Propose formulations of phenylephrine hydrochloride eye drops and

manufacture laboratory scale batches of these.

Characterize the physicochemical properties of the formulations by assessing

appearance, rheology, pH and degradation.

Conduct real time and accelerated stability studies on the formulations; in

accordance with the International Conference for Harmonization (ICH) and

Medicines Control Council guidelines.

Determine the efficacy of antimicrobial preservation.

1.3 Plan of work

In order to achieve the above objectives, a well laid out plan was to be followed.

Literature reviews, on the theory relating to phenylephrine and its prodrugs were

undertaken in an effort to understand the API, eye drops, solutions and product

formulation. Active–excipients compatibility studies were conducted using HPLC and

various eye drops were formulated and manufactured on a laboratory scale. These

underwent stability studies in accordance with International Conference on

Harmonization and Medicine Control Council guidelines. Rheological tests and

efficacy of antimicrobial preservation were demonstrated.

4

2. LITERATURE REVIEW

2.1 Anatomy and physiology of the eye

The human eye provides a challenge to formulators who seek to produce dosage

forms where the API is administered ocularly. This is due to (a) the permeability of

the cornea and (b) the protective operation of the eyelids and lacrimal system. The

operation of the eyelids and lacrimal system rapidly removes materials instilled into

the eye; however, this clearance does not apply to materials that are small in volume

and which are chemically and physiologically compatible with surface tissues

(Hughes, 2004).

The eyes are highly specialized organs of photoreception and are protected by

eyelids and the orbit in which they are placed (Rathore & Nema, 2009). The eye can

be divided into two segments, namely the anterior and posterior segments. The

anterior segment comprises of the cornea, iris, the ciliary body, the anterior chamber

and the posterior chamber while the posterior segment comprises of retina and the

vitreous body as seen in Figure 1 (Ghosh & Jasti, 2005).

Figure 1: Anatomy of the eye (Del Amo & Urtti, 2008).

The unique anatomy, physiology and biochemistry of the eye make it resistant to

foreign substances (Del Amo & Urtti, 2008) this protective mechanism poses a

5

challenge to the formulator who is required to bypass the barriers without causing

damage to the eye (Meqi & Deshpande, 2002). The corneal barrier poses

physiological constraints due to its poor permeability which reduces the absorption of

ophthalmic drugs. The cornea is made up of three membranes; the epithelium, the

endothelium and inner stroma (Chien et al., 1990). The epithelium has tight

junctions that serve as a selective barrier to ion transport, thereby limiting the

diffusion of macromolecules via the paracellular route. The stroma is a highly

lipophilic layer that lies beneath the epithelium, and the more lipophilic a drug is, the

less resistance it will have crossing the stroma (Patel et al., 2010).

The eyelids, conjunctiva, lacrimal systems, cornea–precorneal film and its absorption

are discussed below as they play a major role in the absorption metabolism of eye

drops.

Eyelids: The eyelids have two functions: mechanical protection of the globe (eye)

and creation of an optimum environment for the cornea. The eyelids are lubricated

and kept moist by secretions of the lacrimal glands and specialized cells found in the

bulbar conjunctiva. The antechamber is shaped in a narrow cleft manner directly

over the front of the eyeball, with pocket-like extensions upward and downward. The

pockets are called the superior and inferior fornices (vaults), and the entire space is

called the cul-de-sac. The oval opening between the eyelids is called the palpebral

fissure (Zide, 2006).

Conjunctiva: The conjunctiva is defined as a thin, vascularised mucus membrane

that lines the inner surface of the eyelids and covers the anterior part of the sclera up

to the cornea (Kaur et al., 2003). Its loose attachment permits free movement of the

eyeball. Except for the cornea the conjunctiva is the most exposed portion of the eye

(Hughes, 2004). Uptake of drugs applied topically is greater in the conjunctiva than

in the cornea because the conjunctiva is porous, has a rich blood flow and a large

surface area (Araújo et al., 2009).

Lacrimal system: The conjunctival and lacrimal glands secrete a film of fluid which

covers and lubricates the conjunctival and corneal surfaces. The lacrimal glands

produce tears which are delivered through a number of fine ducts into the

6

conjunctival fornix. A tear is a clear, watery fluid containing salts, glucose, other

organic compounds, approximately 0.7% protein, and the enzyme lysozyme. Small

accessory lacrimal glands are situated in the conjunctival fornices. Their secretion

provides lubrication and cleansing during ordinary conditions and also maintains a

thin fluid film covering the cornea and conjunctiva (the precorneal film). The stability

of the film is maintained through the mucin–protein layer. The sebaceous glands of

the eyelids secrete an oily fluid that prevents overflowing of tears at the lid margin

and reduces evaporation from the exposed surfaces of the eye by spreading over

the tear film (Hughes, 2004).

Blinking helps in replenishing the fluid film by pushing a thin layer of fluid ahead of

the lid margins as they come together. The excess fluid is directed into the lacrimal

lake which is a small, triangular area lying in the angle bound by the innermost

portions of the lids. The skin of the eyelids is the thinnest in the body and folds

easily which permits rapid opening and closing of the palpebral fissures. The eyelids

provide controlled movement such as narrowing of the palpebral fissures in a zipper-

like action from the lateral canthus toward the medial canthus. The transport or

movement of fluid toward the lacrimal lake is aided by the eyelids (Del Amo & Urtti,

2008).

Tears are drained from the lacrimal lake by two small tubes–the lacrimal canaliculi–

which go into the upper part of the nasolacrimal duct, called the lacrimal sac. The

drainage of tears into the nose does not depend only on gravity. Fluid moves along

the lacrimal canaliculi by capillary attraction supported by aspiration caused by

contraction of muscle found in the eyelids. The blinking action causes contraction of

the muscles inducing dilation of the upper part of the lacrimal sac and compression

of its lower portion (Cohen et al., 2006). Tears are aspirated into the sac, which is

collected in its lower part by forcing down the tears through the nasolacrimal duct

toward its opening into the nose. As the muscle relaxes, the lids open. Owing to

muscle relaxation, the upper part of the sac forces fluid into the lower part where it is

simultaneously released from compression. The act of blinking therefore exerts a

suction force-pump action in removing tears from the lacrimal lake as well as

emptying them into the nasal cavity (Cohen et al., 2006). Lacrimation is induced by

reflex action through the stimulation of nerve endings of the cornea or conjunctiva.

7

The reflex could be abolished by anaesthetization of the surface of the eye and by

disorders affecting its nerve components (Cohen et al., 2006).

The cul-de-sac is free of pathogenic organisms and is sterile. The sterility is due to

the action of lysozyme, in the tears, which destroys saprophytic organisms but has

little action against pathogens. Certain diseases cause the lacrimal gland, to undergo

involution, resulting in scanty lacrimal fluid production and a change in the

conjunctival glands also leads to changes in the amount of the secretions. This may

lead to symptoms of dryness, burning and general discomfort and may interfere with

visual acuity (Kaur & Kanwar, 2002).

Precorneal film: The precorneal film is composed of a thin outer lipid layer, a thicker

middle aqueous layer, and a thin inner mucoid layer. It is renewed during each blink,

and when blinking is suppressed, either by drugs or by mechanical means, it dries in

patches. The precorneal film is unaffected by the addition of concentrations of up to

2% sodium chloride into the conjunctiva. The precorneal film, which is part of the

tear fluid, maintains the cornea’s moist surfaces and its functionality depends on the

condition of the corneal epithelium. A pH below four or above nine causes

derangement of the film (Grosvenor, 2007).

Cornea: The cornea has a thickness of 0.5 to 1 mm and consists mainly of the

following structures (from the front backward): Corneal epithelium, subtantia propia

(stroma), corneal endothelium. The cornea is transparent to visible light due to the

special laminar arrangement of the cells and fibres and because of the absence of

blood vessels (Chien & Schoenwald, 1990). Cloudiness of the cornea may be due to

several factors including excess pressure in the eyeball as in glaucoma, and scar

tissue due to injury, infection, deficiency of oxygen or excess hydration such as may

occur during the wearing of improperly–fitted contact lenses. Trauma to the cornea

usually heals as an opaque patch that can permanently impair vision unless it is

located in the periphery of the cornea (Kaur et al., 2003).

The outer surface of the cornea is most responsible for the refraction of light

whereby the index of refraction changes from that of air (1.00) to that of precorneal

substance (1.38). Alteration in its shape or transparency interferes with the formation

8

of a clear image; therefore any pathological process, however little, may seriously

interfere with the resolving power or visual acuity of the eye (Lang et al., 2005).

The normal cornea possesses no blood vessels except at the corneoscleral junction.

The cornea derives its nutrition from the aqueous humour by diffusion and has

certain permeable characteristics. It also receives nourishment from the fluid

circulating through the chambers of the eye and from the air. The corneal epithelium

provides an efficient barrier against bacterial invasions due to its poor blood supply.

Foreign bodies that either scratch the cornea or lodge or become embedded in the

cornea are of serious concern, as the presence of any of these could play a role in

permitting pathogenic bacteria to gain access to the cornea. Trauma plays an

important part in most of the infectious diseases of the cornea that occurs

exogenously (Ghosh & Jasti, 2005).

Corneal absorption: The cornea is a membrane having both hydrophilic and

lipophilic layers. It is composed of three general layers: the lipid–rich epithelium, the

lipid-poor stroma, and the lipid rich endothelium. The combined lipid content of the

corneal epithelium and the corneal endothelium approximately 100 times more than

the lipid content of the corneal stroma. Modern ocular drug delivery system designs

are based on an understanding of drug deposition pathways within the relative lipid

content of these layers and their overall ocular pharmacokinetic/pharmacodynamic

profile (Curtin & Cormican, 2003). Only drugs having both lipid and hydrophilic

properties have the greatest corneal penetration. Highly water–soluble drugs

penetrate less readily. For example, highly water–soluble steroid phosphate esters

penetrate the cornea poorly and for better penetration to be attained a poorly soluble

but more lipophilic steroid alcohol is used (Holladay, 2006).

Eye drops penetrate the eye primarily through the cornea. Typical drug penetraton

occurs as follows:

a) The drug molecule as a free base and the salt will be in an equilibrium

depending on the drugs physicochemical characteristics and the pH.

9

b) If the formulation is slightly acidic at the moment of instillation, the acidity is

neutralised by the lacrimal fluid which converts it rapidly to the physiological

pH range ( pH 7.4).

c) There will be sufficient free base to begin penetration of the corneal

epithelium at this pH. An undissociated drug molecule will penetrate the

stroma, epithelium, and endothelium because it is water–soluble.

d) The dissociated drug leaves the endothelium for the aqueous humour, as it

can readily diffuse to the iris, the ciliary body, and the site of the

pharmacological actions (Nanjawade et al., 2007).

Martini and colleagues (1997) discussed the implications of the mechanism of

precorneal drug loss in the design of ocular drug-delivery systems, including the

effect of instilled drug volume on aqueous humour concentrations and the amount of

drug available for systemic absorption. Ideally, smaller volumes permit more drugs

to be absorbed. For a given instilled concentration the opposite is true; however, a

smaller volume instilled remains more efficient (Friedrich et al., 1993).

There are two types of corneal penetrations: transcorneal and non-corneal

absorption (Nanjawade et al., 2007). Lang and colleagues (2005) stated that the

transcorneal route of absorption of a drug into the eye is the route most effective in

bringing a given drug to the anterior portion of the eye. This is enhanced by the

water-lipid gradient found in the cornea.

Drugs penetrate the corneal epithelium with the use of transcellular or paracellular

pathways which are both types of transcorneal penetration. Transcellular and

paracellular pathways are used by both lipophilic and hydrophilic drugs; these

pathways involve passive diffusion or modified diffusion through intercellular spaces

and most topically–applied drugs diffuse passively along their concentration gradient.

However, the stereospecific carrier–mediated transport system is used by certain

drugs as their mode of transport such as l-lysine which makes use of the Na+ –K+ –

ATPase pump as medium of transport in the cornea (Urtti, 2006). Physicochemical

properties of drugs have properties such as lipophilicity, solubility, molecular size

and shape, charge and degree of ionization affects the route and rate of permeation

in the cornea (Järvinen et al., 1995). The hydrophilic drugs are rate–limited by the

10

lipophilic corneal epithelium, while lipophilic drugs partitioning from the epithelium to

the hydrophilic stroma are rate limiting (Järvinen et al., 1995).

Non-corneal absorption pathways involve the penetration of drug across the bulbar

conjunctiva and sclera into the uveal tract and vitreous humour. The conjunctiva and

sclera provide a route for large hydrophilic molecules for example inulin and p-

aminoclonidine (Nanjawade et al., 2007; Lang et al., 2005).

The cornea can be penetrated by small ions through extracellular spaces to a

measurable degree. The diameter of the largest particles that can pass across the

cellular layers is in the range of 10 to 25 Å. Permeation of instilled drugs can also be

reduced by protein binding in the tear fluid and metabolic degradation by enzymes.

There are numerous drug metabolizing enzymes present in the cornea like

esterases, peptidases, reductases and cytochrome P (CYP) family enzymes among

others (Gaynes & Fiscella, 1996; Mashkevisch, 2007). Enzymes help in drug

detoxification and metabolism. These enzymes also maintain ocular homeostasis by

preventing environmental and systemic injuries (Mashkevisch, 2007). Martini and

colleagues (1997) concluded that the following are responsible for precorneal drug

loss in a descending order: drainage vasodilation > nonconjunctival loss > induced

lacrimation conjunctival absorption > normal tear turnover.

2.2 Pathophysiology of the eye

Ocular manifestation of allergy in which the body produces hypersensitivity to

normally harmless substances (allergens) is classified to be an allergic eye disease.

Allergy prevalence in Europe is between 15 and 20%; by 2015 the rates are

estimated to increase to 50% which is about 20% more (Bilkhu et al., 2011).

Improved hygiene practices and increased antibiotic use by modern day lifestyle in

addition to environmental factors such as increased air pollution, climate change and

increased planting and importation of allergenic plant species have been reported to

increase the allergy prevalence, although genetics plays an important role in

susceptibility (Bilkhu et al., 2011).

11

Seasonal allergic conjunctivitis (SAC) is the most common form of allergic eye

disease that constitutes of 90% of the allergy cases with perennial allergic

conjunctivitis at 5% (Bielory, 2008). SAC is most frequently caused by grass, tree

and weed pollens and outdoor moulds which peak at different times of the year

(Bielory, 2008; Chigbu, 2009), often as part of seasonal rhinoconjunctivitis (hay

fever). However, PAC occurs year round due to house dust mites, animal dander,

insects and indoor moulds (Chigbu, 2009). The signs and symptoms of SAC develop

gradually but sometimes it can develop suddenly in contact with an offending

allergen (Bielory, 2000 & Bielory 2008). Some of the signs and symptoms of SAC

includes itching, tearing, eyelid oedema and conjunctival hyperaemia, chemosis and

papillary reaction in which the severity often varies with pollen counts (Cox, 2007).

PAC and SAC have similar signs and symptoms but differ as PAC is milder and

chronic in nature and may have seasonal exacerbations (Bielory, 2008 & Buckley,

1998). The impact of allergic eye disease on the quality of life can be profound even

though the signs of symptoms of SAC and PAC are relatively mild (Bielory, 2006). It

affects daily activities, productivity at work, school performance and the

economy (Pitt et al., 2004; Smith et al., 2005; Palmares et al., 2010). The severities

of presentation limits the complications of SAC and are linked to steroid use in cases

refractory to conventional treatment (Hingorani and Lightman, 1995; Joss & Craig,

1998; Bielory et al., 2010). The treatments provided for SAC are mainly for

preventing and alleviating symptoms rather than cure due to the fact that no cure has

been found or produced for SAC or any allergy. However, immunotherapy shows

promise (Bielory, 2008).

The pathophysiological mechanism of acute ocular allergy involves acute antibody

(immunoglobulin E (IgE))-mediated mast cell degranulation and minimal presence of

migratory inflammatory cells. In chronic ocular allergy, the pathophysiology consists

of persistent activation of mast cells and eosinoiphil–T–lymphocyte-mediated

delayed-type hypersensitivity (DTH) response. Antigen-specific antibodies, such as

IgG and IgE contribute to the pathogenesis of Th2-mediated diseases including

allergies. IgE and IgG receptors are expressed on mast cells and as such IgE and

IgG may participate in mast cell activation and mediator release. Antibody (IgE or

IgG, particularly of the IgG1 isotype–mediated–mast cell activation results in

degranulation, with the release of pre-formed and newly formed pro-inflammatory

12

mediators as well as the secretion of chemokines and cytokines (Tkaczyk et al.,

2002).

Both SAC and PAC are IgE-mast cell mediated hypersensitivity reactions, divided

into two phases with the mast cell playing a central role (Bacon et al., 2000; Choi &

Bielory 2008). The reaction involves a very complex series of immunological events

coordinated by various mediators initiated by an allergen (Ono & Abelson, 2005 ;

Hodges & Kean–Myers, 2007) An allergen such as pollen reacts with specific IgE

antibodies bound to a sensitised mast cell, triggering cross linkage of the IgE

molecules and an influx of calcium ions into the mast cell. This causes the mast cell

to degranulate and release preformed inflammatory mediators such as histamine

which cause the signs and symptoms associated with the early phase response in

sensitised individuals. The early phase response is immediate and lasts clinically for

20–30 minutes (Leonardi et al., 2008).

With increasing knowledge of the pathophysiology/underlying mechanisms of allergic

eye disease, there has been a rapid and large increase in the number of

pharmacological anti–allergic medications available for treatment. Several authors

have pointed out that ocular allergies have been under-diagnosed and under-treated,

in particular seasonal allergic conjunctivitis where the ocular symptoms fall under the

umbrella of seasonal hay fever which may underestimate its true prevalence (Cox,

2007; Bielory, 2008; Berdy & Berdy, 2009). A recent study by Wolffsohn and

colleagues (2011) highlighted the current poor management of ocular allergies,

where patients often self-medicate and rarely undergo an ophthalmic examination.

Due to the increasing prevalence of allergy, allergic eye disease is becoming more

common and combined with its impact on the quality of life and inadequate

management, it is important for practitioners to identify these patients in order to

manage them appropriately.

Mast cell degranulaion also initiates a series of cellular and extracellular events

which lead to the late phase response, including production of prostaglandins,

thromboxanes and leukotrienes derived from arachidonic acid. Mast cells also

release cytokines and chemotactic factors which induce the production of IgE from

B-cells, enhance production of Th2-lymphocytes, attract eosinophils and activate

13

vascular endothelial corneal and conjunctival cells to release chemokines and

adhesion molecules. The chemokines and adhesion molecules mediate the

infiltration of eosinophils, basophils, neutrophils and Th2-lymphocytes to the site of

inflammation and coupled with the newly formed mediators and sustained mast cell

activation they result in the late phase response (Stahl et al., 2002). This may occur

3–12 hours after the initial reaction and symptoms can continue up to 24 hours. The

year round symptoms associated with PAC are the result of chronic mast cell

activation and Th2-lymphocyte infiltration (Bonini et al., 1989).

The most important and most effective step in treating allergic eye disease is

avoiding the offending allergen to prevent the hypersensitivity reaction from being

triggered. This necessitates the identification of the offending allergen and complete

avoidance is not always possible (Friedlaender, 2001). In SAC a detailed history is

essential as knowledge of the period of time of year symptoms occur can allow

identification using a pollen calendar to some extent but peak levels of common

causative pollens often overlap. However, effective measures for allergen avoidance

in SAC and PAC are based upon control of the environment. Given that pollens are

the main cause of SAC, preventative measures include limiting outdoor activity

during the symptomatic period, closing windows and using air conditioning when in a

car or indoors, avoid touching/rubbing eyes after being outdoors, wash hands after

being outdoors and wearing close fitting or wrap around style sunglasses when

outdoors (Veys, 2004). As PAC can affect the patient all year round, more thorough

avoidance measures are necessary. Dust mite levels in the home can be reduced by

using and regularly replacing protective pillow, mattress and duvet covers; washing

bedding regularly at least at 60 °C; vacuum and damp dust entire house on weekly

basis; reduce humidity to between 35 and 50% and remove or regularly clean

carpets, upholstery, curtains and any other areas that gather dust (Gotzche &

Johansen, 2008; Scheikh et al., 2010). Animal dander can be reduced by eliminating

all pets/animals from the home or keep them outdoors; regular vacuuming;

minimising exposure to areas that gather animal dander; avoid touching animals;

washing hands and avoid eye touching/rubbing after contact with animals; and

washing all clothes that have come into contact with animals (Eggleston & Wood,

1992; Bush, 2008).

14

Despite implementing these avoidance measures, complete avoidance is not always

possible so use of anti–allergic medication may become necessary to prevent and

alleviate symptoms. With increased knowledge of the pathophysiology of the

hypersensitivity reaction in SAC and PAC over the years, there has been a rapid

increase in the number of anti-allergic medications that target the immunological

cells and inflammatory mediators involved in the allergic expression. Ophthalmic

anti-allergic medications include topical mast cell stabilisers, antihistamines,

vasoconstrictors (phenylephrine hydrochloride), antihistamine–vasoconstrictor

combinations and dual action agents with mast cell stabilising and antihistaminic

properties (Schultz, 2006).

Studies on the ocular use of phenylephrine hydrochloride started in 1933 and were

first reported in 1936 (Greaves et al., 1992). The present research or study is based

on the insight into phenylephrine hydrochloride and its properties.

2.3 Phenylephrine hydrochloride and its ocular uses

2.3.1 Phenylephrine hydrochloride

Phenylephrine is a sympathomimetic drug, used to relieve pain associated with

complicated uveitis, allergic conditions, reduce posterior synechiae formation, and

induce mydriasis, and as a topical spray into the ear to reduce the pressure in the

Eustachsian tube (Johnson et al., 2008; Ahmed and Amin, 2007). Some of the

commonly found salts are: phenylephrine hydrochloride; phenylephrine acid tartrate;

phenylephrine bitartrate; phenylephrine tannate and phenylephrine tartrate

(Sweetman, 2002). Phenylephrine oxazolidine, which is an ester of phenylephrine,

has been found to provide longer retention time with lower plasma levels, thereby

reducing chances of side effects like increased blood pressure. There is no salt form

for phenylephrine oxazolidine (Rautio et al., 2008).

Phenylephrine hydrochloride was used in this study due to its pharmaceutical

stability and availability (Trommer et al., 2010). The following discussion will

therefore be limited to phenylephrine hydrochloride.

15

2.3.2 Pharmacological actions and uses

Phenylephrine, or 3-(1–hydroxyl–2–methylamino-ethyl) phenol (Trommer et al.,

2010) is a synthetic, imidazole-derivative, completely used in the optically active form

(Pandey et al., 2003). Figure 2 shows the structure and available salts of

phenylephrine in the pharmaceutical industry.

Figure 2: Structure of Phenylephrine, its base and salts (Trommer et al., 2010).

Ornato and Peberdy (2005) showed that phenylephrine hydrochloride solution could

be used in place of epinephrine to increase regional cerebral blood flow following a

prolonged cardiac arrest during cardiopulmonary resuscitation.

2.3.3 Mechanism of action

Vasoconstriction of the arterioles of the conjunctiva is due to PE’s action as a potent

direct-acting alpha-adrenergic stimulator with weak beta-adrenergic activity (Leikin &

Paloucek, 2007). It causes contraction by activating the dilator muscle of the pupil

(Leikin & Paloucek, 2007). It also stimulates alpha-adrenergic receptors and

intracellular acetylate cyclase are inhibited by phenylephrine, the intracellular

adenylate cyclase then inhibits the production of cAMP (Donnelly, 2009). This

inhibition of cAMP causes arterial and venous constriction in the conjunctiva thereby

decreasing the blood flow and mucosal oedema caused by allergic responses

(Donnelly, 2009).

16

2.3.4 Pharmacokinetics

The protective mechanisms that are present in the eye to shield the visual pathway

from foreign chemicals make it hard for drug to be delivered to the eye. These

mechanisms have led to the development of optimized ophthalmic drug delivery

systems that are based on the understanding of the drug disposition pathways in the

eye and the ophthalmic pharmacokinetic profile (Worakul & Robinson, 1997).

Phenylephrine hydrochloride is administered topically to avoid extensive first pass

metabolism and to reduce systemic side effects (Aschenbrenner & Venable, 2008).

Peak ocular concentrations of 1.2% to 5% are achieved after a single dose between

20 to 60 minutes. Maximal dilation of the pupil occurs within 60–90 minutes and the

effect lasts 5–7 hours (Rossiter, 2010).

Phenylephrine has a half-life of a few minutes when circulating in the blood. It can be

degraded either through methylation by COMT or by deamination by MAO in the

blood stream (Flancbaum et al., 1997). The dehydrated form of phenylephrine

hydrochloride is a major degradation derivative during pharmaceutical processing

and stability determination (Trommer et al., 2010).

2.3.5 Adverse effects

Phenylephrine produces systemic adverse effects such as hypertension,

subarachnoid haemorrhage, ventricular arrhythmias and myocardial infarction,

trembling, headache and sweating (Dipiro et al., 2002).

The day after administration, rebound miosis has a possibility of occurring

particularly for older patients (Aschenbrenner & Venable, 2008).

Eye drops containing 10% of API may have profound effects on the cardiovascular

system, and the risk is smaller with 2.5% products, which are said to be equally

effective as mydriatics (Rossiter, 2010). The hypertensive effects of phenylephrine

may be treated with an alpha–adrenoceptor blocking agent such as phentolamine

mesylate, 5 to 10 mg intravenously which should be repeated as necessary (Beyene

& Maren, 2004).

17

2.3.6 Drug interactions

Phenylephrine may produce severe hypertension and cardiac arrhythmias when

used concurrently with beta blockers or other antihypertensive (for example

reserpine) and, tricyclic antidepressants. Mono amine oxidase inhibitors increase

pressor effects of phenylephrine which potentiates the risk of hypertensive crisis

(Pray, 2006).

2.3.7 Bioavailability

The average human being has a tear volume of 7 µL while the cul-de-sac has a

maximum capacity of about 30 µL. Many ophthalmic formulations range from 50 to

75 µL in volume; however, volumes in excess of 50 µL are probably unable to enter

the cul-de-sac (Greaves et al., 1992). Systemic absorption occurs through solution

drainage into the nose, which causes the loss of an instilled drug as shown in Figure

3 (Jarvinen et al., 1995).

Figure 3: Diagram of Ocular Absorption (Nanjawade et al., 2007).

The equation describing the above processes in terms of drug concentration is:

Drug in tear fluid

Ocular absorption (5% of the dose)

Systemic absorption (50-100% of the dose) -Conjunctiva of the eyes -Nose Minor routes: -Larcrimal drainage system -pharynx GI-tract -Skin at the cheek and lids -Aqueous humour -Inner ocular tissues

Corneal route

- 1 route - Small, lipophilic drugs

Conjunctival and Scleral route -Large, hydrophilic drugs

Aqueous humour

Ocular tissues

18

Where F is the fraction of dose absorbed, D is the dose, k and K are absorption and

elimination rate constants, respectively, and V, is the apparent volume of distribution

(Lee & Robinson, 1979; Worakul &Robinson, 1997; Lee & Robinson, 1986).

An ideal eye drop preparation possesses a high concentration of drug in a minimum

drop volume. Martini and partners (1997) reported that equal tear-film concentrations

were obtained whether either 5 µL of 1.61 x 1010 M or 25 µL of 1.0 x 1010 M

pilocarpine nitrate solution were administered. The higher bioavailability of the 5 µL

solution was due to decreased drainage loss. However, there is a practical limitation

to the minimum dosage volume used. Dropper configuration that delivers small

volumes poses difficulties in its design and patients often cannot sense or detect the

administration of volumes in the 5.0 to 7.5 µL dose volume ranges (Martini et al.,

1997).

2.3.7.1 Reasons for poor ocular bioavailability

The most common method for the administration of therapeutic treatment for ocular

diseases is the topical delivery of eye drops. The rapid elimination of drugs from the

precorneal lachrymal fluid by solution drainage, lachrymation, and poor absorption

by the conjunctiva creates problems for topical delivery (Bartlett & Jaanus, 2008).

The eye has the ability to permanently maintain its residence volume at 7–10 µL due

to high drainage rate. The volumes instilled with eye drops range from 20–50 µL,

therefore the residence time of solutions is limited to a few minutes, causing the

overall absorption of a topically applied drug to be limited to 1–10% (Felt et al.,

1999).

19

2.3.7.2 Strategies for improving drug availability in ocular adminstration

There are three main ways to maximise the systemic bioavailability of drugs

administered ocularly, these are:

Increase ocular residence time.

Increase ocular absorption.

Alter the drug structure to change physicochemical properties (Lang et al.,

2005).

2.3.7.2.1 Increasing ocular residence time

Lacrimal secretions and drainage systems act to remove foreign bodies and

substances from the corneal epithelium as quickly as possible. In order to increase

residence time, or delay clearance, drops should be instilled in the anterior segment

of the ocular cavity. Products can also be formulated with polymers such as

methylcellulose, hydroxypropyl methylcellulose or polyacrylic acid (Carbopol), which

increases the viscosity of the formulation and act as bio–adhesives with the ocular

tissue (Patel et al., 2010).

2.3.7.2.2 Increasing ocular absorption

Enhancers act by increasing the rate at which drugs penetrate the epithelium. They

alter the structure of the epithelial cells to increase absorption; this is achieved

without destruction of the cells. Some examples are dimethyl sulfoxide,

decamethonium, EDTA and glycocholate. Ideal absorption enhancers should

possess the following qualities:

They should provide an effective increase in the absorption of the drug.

Should not cause permanent damage.

Must be non–irritant.

Must be effective in small quantities.

Effect should be fully reversible and temporary.

Should not have a lag effect when absorption is required.

20

Should be stable (Saettone et al., 1996; Patel et al., 2010).

2.3.7.2.3 Altering drug structure

Modifications to a drug molecule are usually done to achieve certain

physicochemical properties in order to, amongst others, increase aqueous solubility

and improve partitioning. Caution should be applied in drug modification as it could

pose the risk of changing a drug’s therapeutic and toxicological profile. Subsequent

changes in salts or molecules could result in increased costs and lengthy

investigations due to regulatory requirements. Some factors that have been found to

influence drug absorption are its weight and pH (Krishnamoorthy & Mitra 1998).

Molecular size and weight: Ocular absorption of small compounds, approximately

100 Daltons, is higher, (around 80%), when compared to oral absorption. This

however reduces markedly as the molecular weight increases. There is a restriction

to the molecular weights that can pass through pathways and channels (molecular

weight cut–off) as only molecules that are smaller than the channels can diffuse

through them (Akiba et al., 1993).

The effect of pH and the partition coefficient: The partition coefficients of drugs

are dependent upon environmental pH which affects the ionization of drugs (local pH

can be modified by ocular formulation). The rate of absorption is increased when the

drug or substance is un–ionized. The movement of ionizable drugs through

membranes depends on the chemical equilibrium between ionized and unionized

drug in the eye drop and the lacrimal fluid (Geroski & Edelhauser, 2000). The ionized

form does not easily penetrate the lipid membrane compared to the unionized

molecule. The partitioning of ionized molecule does not only depend on the degree

of ionization but also on the charge of the molecules (Liaw et al., 1992). The corneal

epithelium is negatively charged (isoelectric point is pI 3.2), and as a result,

hydrophilic–charged cationic substances permeate easily through the cornea than

anionic compounds (Liaw et al., 1992). Compounds below pI 3.2 are negatively-

charged and pass through the cornea making the pH too acidic, and irritating for

clinical use. Consequently, in practice, charge–discriminating effects of the corneal

21

epithelium decrease the absorption of negatively charged compounds (Rojanasakul

& Robinson, 1989; Conroy & Maren, 1995; Taylor, 2002; Lee & Robinson, 2009;

Hecht, 2000).

2.3.8 Polymorphism and pseudomorphism of phenylephrine hydrochloride

A polymorph is a solid substance with the ability to crystallize in at least two different

crystal structures that produce distinct crystal species. Polymorphism is important in

the drug development process as it can affect drug dissolution, chemical stability, as

well as drug bioavailability (Yoshihashi et al., 2002). Polymorphs are the collection of

different crystal structures that can exist for a single chemical entity and its hydrates.

The difference between a solvate and polymorph is that a solvate contains different

quantities of solvent trapped within the crystal structure (as is the case for

MgSO4.7H2O and MgSO4.10H2O) and polymorphism is when there is no solvent

trapped in the crystal structure and the same chemical species are found in various

crystal structures. Solvates or false polymorphs are called pseudopolymorphs.

Pseudopolymorphs can be obtained by changing the recrystallizing solvent.

Common solvents used to induce polymorphic change are water, methanol, ethanol,

and acetone and chloroform (Lee et al., 2006). Phenylephrine hydrochloride does

not exhibit polymorph or solvate forms, its chemical stability will therefore not be

hampered when dissolved in solvents like water and glycerol (Yoshihashi et al.,

2002).

2.4 Ophthalmic formulations

Eye drops are sterile products, free from foreign particles, compounded and

packaged for ocular drug delivery. Eye drops can be prepared as single or multidose

products (Van Santvliet & Ludwig, 2004).

Compared to the buccal, nasal, rectal, vaginal or dermal routes, eye drops are easier

to use, non-invasive, accurate and less expensive for systemic delivery of drugs

(Pillion et al., 1994). The ocular route helps eliminate painful parenteral injections or

gastro-intestinal degradation (Chiou et al., 1991 and Pillion et al., 1994).

22

Absorption of the eye drops across the mucosa in the nasal cavity that is near to the

conjunctival sac helps eye drops with active pharmaceutical ingredients reach the

bloodstream (Winfield et al., 2009). Physicochemical drug properties, such as

lipophilicity, solubility, molecular size and shape (Huang et al., 1989; Liaw &

Robinson, 1992), charge (Rojanasakul et al., 1992; Liaw et al., 1992) and degree of

ionization (Brechu & Maren, 1993) affect the route and rate of permeation in the

cornea.

Drugs can be absorbed through the naso-lacrymal system and reach systemic

circulation without passing metabolism by the liver only if there is (a) drainage loss of

topically-applied solutions,(b) increase in bioavailability of the instilled drug (Saettone

et al., 1984), and (c) an increase in viscosity (Flach, 2002). Mild to severe side

effects may therefore be observed. The phenomenon has led to the reduction in the

size of eye drops (Saettone et al., 1984; Ludwig & Van Ooteghem, 1989; Salminen,

1990; Flach, 2002).

2.4.1 Eye drops as an ophthalmic dosage form

Liquid formulations (eye drops and eye lotions), semisolids (creams, ointment and

gels), solids (biodegradable implants) are various ophthalmic dosage forms used to

deliver therapeutic agents topically (Kaur & Kanwar, 2002). Eye drops are

conventional dosage forms that account for 90% of the currently accessible

ophthalmic formulations. Eye drop solutions are common dosage form as they offer

advantages like administrations, easy preparation and low costs. Eye drops also

have some disadvantages like short contact time with ophthalmic surface and

nasolacrimal drainage which causes poor bioavailability of the drug (Ali &

Lehmussaari, 2006). Ophthalmic delivery systems are being investigated in order to

increase the corneal permeability and prolong the contact time with the ocular

surface through the addition of viscosity modifying agents such as hydroxypropyl

methylcellulose, carboxy methylcellulose sodium and other cellulose derivatives

(Gad, 2008).

Suspensions are defined as the dispersion of finely divided, relatively insoluble drug

substances in aqueous vehicle containing suitable suspending and dispersing

agents (Hecht, 2000). Due to the tendency of particles being retained in the cul-de-

23

sac, the contact time and duration of action of a suspension could theoretically

exceed that of a solution. There are difficulties associated with suspension dosage

forms such as dosage uniformity, crystal growth and polymorphism. Dosage

uniformity requires a brisk “shake well” approach to distribute the suspended drug.

This can contribute towards poor patient compliance as significant numbers of

patients do not adhere to instructions (shake well before use). Polymorphism can

occur during storage resulting in an increased or decreased solubility whereby

changes are reflected in increased or decreased bioavailability (Hecht, 2000).

Ointments offer longer contact time, greater total drug bioavailability albeit with

slower onset and time to peak absorption. Ointments interfere with vision and are

usually restricted to bedtime use. They remain a popular paediatric dosage form and

for postoperative use (Gad, 2008).

Solutions are the preferred ophthalmic dosage forms for treatment of conditions such

as uveitis (Ali & Lehmussaari, 2006). These forms are preferred because:

a) All the ingredients are completely in solution with uniformity.

b) There is reduced systemic toxicity.

c) Rapid onset of action is achieved.

d) The required dose over time is decreased.

e) There is little physical interference with vision.

f) It offers a convenient mode of administration (Kaur et al., 2003: Ghosh &

Jasti, 2005).

Limited bioavailability due to short contact time between the eye and the active

pharmaceutical ingredients is one shortcoming of solutions. The short contact time

between the active pharmaceutical ingredient and the eye surface can be increased

by the inclusion of a viscosity-modifying agent such as methylcellulose. The optimum

level of drug retention and visual comfort is within the viscosity range of 0.15 to 0.25

Pa·s (Lang et al., 2005).

There are factors to be considered in the preparation of eye drop solutions. These

include sterility, clarity, buffer, buffer capacity and pH, tonicity, viscosity, stability,

comfort, additives, particle size, packaging and preservatives. These factors are

24

interrelated and assessed collectively in the preparation of an eye drop product. The

buffer system is considered along with tonicity and comfort. Stability of the eye drop

product depends on pH, buffer system, and packaging. Viscosity-modifying agents

which may or may not be present should not affect the therapeutic effectiveness of

the active ingredient (Lang et al., 2005).

The pH and buffer capacity is a compromise between stability of the drug and

comfort in the eye. Optimum patient comfort is found at the pH of the tear fluid, or at

7.4 pH, while optimum stability for many drugs is lower than 5 pH. Buffers are

therefore needed to maintain the desired pH for drug stability and allow it to be

altered to 7.4 immediately after instillation in the eye (Aulton, 2002).

Table 1: Conventional dosage forms and usage (Lang, 1995).

Formulation Number %

Gels 2 0.7

Injectables 11 3.8

Inserts 11 3.8

Ointments 50 17.4

Orals 9 3.1

Solutions 179 62.4

Suspensions 25 8.7

2.4.2 Eye drop formulation characteristics

2.4.2.1 Clarity

Eye drop solutions should be clear which is achieved by filtration. Filtration

equipment, which has a pore size of 0.45 µm, must be sterile particle free so that

particulate matter is not found in the solution by equipment designed to remove it.

Sterile techniques must be performed in clean surroundings: and using laminar-flow

hoods and proper garments will aid in preparing solutions free from foreign particles.

With sophisticated machines clarity and sterility can be achieved in the same

filtration step. It is important to note that solution clarity is equally dependent on the

cleanliness of the intended container and closure. Both container and its closure

must be sterile. This means that the container or closure must not contribute

25

particles to the solution during prolonged contact such as shelf-life storage (Michael

& Richards, 2009).

2.4.2.2 Stability, pH and buffer systems

Stability is not only the chemical stability of a single product component but the total

product. A well-planned stability programme will consider and evaluate the chemical

stability of the active ingredient’s preservative efficacy against selected test

organisms, and test the adequacy of the package over time (McDonnell, 2007).

The stability of a drug in an eye drop product depends on the physicochemical

nature of the drug substance, pH, method of preparation (temperature exposure),

additives, and type of packaging. Initially eye drop solutions had a short shelf life

however; with recent advancement in technology the stability of eye drop products is

expressed in terms of years usually between two-three years (McDonnell, 2007).

Ideally, the prepared eye drop should be formulated at a pH equivalent to the tear

fluid value of pH 7.4. The majorities of active ingredients used in eye drops are salts

of weak bases and are stable at acidic pH. The acidic pH selected should therefore

be maintained throughout the product’s shelf life, however, if the buffer capacity is

sufficient to resist adjustment by tear fluid, and the overall eye pH remains acidic for

a considerable period, stinging and discomfort may result (Florence & Attwood,

2006). The buffer capacity should thus be adequate for stability but minimized, as far

as possible, to allow the overall pH of the tear fluid to be disrupted only momentarily

(Florence & Attwood, 2006). Common buffering agents found in eye drop

formulations are sodium citrate dihydrate and boric acid (Florence & Attwood, 2006).

Sodium citrate dihydrate is used primarily to adjust the pH of solutions. It is used at

concentrations of 0.1–2.0% in ophthalmic products. It is a stable material and can be

easily sterilized by autoclaving (Amidon, 2006). The bulk material should be stored in

a well–closed container protected from light, in a cool, dry, place (Harwood, 2006).

26

Boric acid is odourless, colourless and greasy to the touch. It has weak bacteriostatic

and fungistatic properties. It is commonly used as a buffer and antimicrobial in eye

drops at concentrations of 1.22%. Boric acid volatilizes in steam (Stewart, 2004).

Oxygen sensitivity is an important factor to be considered as certain active

ingredients may require the inclusion of an antioxidant (Ansel et al., 2011; Sutton et

al., 1998 and Mitra, 2009).

Phenylephrine hydrochloride is stable at a pH range of 3.5–8. It shows signs of

degradation by changing its clear colour to yellow or brown, when dissolved in

aqueous solutions (Lang et al., 2005).

2.4.2.3 Tonicity

Tonicity refers to the osmotic pressure exerted by salts in aqueous solution. An eye

drop solution is isotonic with another solution when the magnitudes of the colligative

properties of the solutions are equal. An eye drop solution is considered isotonic

when its tonicity is equal to that of a 0.9% sodium chloride solution. The calculation

of tonicity at one time was stressed rather heavily to the detriment of other factors

such as sterility and stability. In actuality the eye is much more tolerant of tonicity

variations than it was formerly thought. The eye tolerates solutions equivalent to a

range of 0.5 to 1.8% sodium chloride (Ansel et al., 2011).

Glycerol is used in ophthalmic pharmaceutical formulations as a tonicity modifying

agent with an antimicrobial and viscosity-modifying functions when used at

concentrations of >20% and 0.5–3% respectively. Glycerol is hygroscopic and not

prone to oxidation by the atmosphere under ordinary storage conditions, but

decomposes on heating, with the evolution of toxic acrolein. Mixtures of glycerol

with water, ethanol, and propylene glycol are chemically stable. Black discolouration

of glycerol occurs in the presence of light on contact with zinc oxide or basic bismuth

nitrate (Price, 2006).

27

2.4.2.4 Viscosity

Medicated eye drops usually have poor bioavailability due to the barrier created by

the precorneal area. The site of absorption is cleared rapidly by protective

mechanisms such as blinking and nasolacrimal drainage (Ali & Lehmussaari, 2006).

This necessitates frequent instillation, increasing side effects associated with the

active pharmaceutical ingredient (Di Colo et al., 2009). There is need therefore to

increase API residence in the eye by increasing or adding the polymer that prolongs

drug contact time with the ocular surface (Wilson, 2004).

Substances such as methylcellulose, polyvinyl alcohol, and hydroxypropyl

methylcellulose can be added to increase viscosity and prolong contact time in the

eye which enhances drug absorption and activity. Viscosity ranging from 0.15 to 0.25

Pa·s significantly improves contact time in the eye. Results tend to plateau beyond

the 0.25 Pa·s as higher viscosity values offer no significant advantage (Winfield &

Richards, 2004).

Hydroxypropyl methylcellulose is a widely used thickening agent in topical

pharmaceutical formulation especially for ophthalmic formulations (Romanelli et al.,

1994) Compared with methylcellulose; hydroxypropyl methylcellulose produces

solutions of greater clarity, with fewer undispersed fibres present, thus making it

preferable for formulations for ophthalmic use. Its concentration is between 0.45-

1.0%w/w (Harwood, 2006).

For an aqueous solution to be prepared, hydroxypropyl methylcellulose should be

dispersed and thoroughly hydrated in about 20–30% of the required amount of water

(Harwood, 2006).The water used should be vigorously stirred and heated to between

80–90 °C and the remaining hydroxypropyl methylcellulose added. Cold water should

be added to produce the required volume (Harwood, 2006).

Aqueous solutions of HPMC are enzyme-resistant, and provide viscosity stability

during long-term storage. However, aqueous solutions are liable to microbial

spoilage and should be preserved with an antimicrobial preservative. When used as

a viscosity-increasing agent in ophthalmic solutions, benzalkonium chloride is

28

commonly used for antimicrobial protection. Aqueous solutions may be sterilized by

autoclaving; the coagulated polymer must be redispersed on cooling by shaking

(Harwood, 2006).

Carboxymethylcellulose sodium is widely used as a topical pharmaceutical

formulation primarily for its viscosity-modifying properties and its concentrations

range between 3–6%. Viscous aqueous solutions are used to suspend powders

intended for topical administration (Parsons, 2006).

Carboxymethylcellulose sodium is a stable hygroscopic material. Under high

humidity conditions carboxymethylcellulose sodium can absorb a large quantity of

water (> 50%). Aqueous solutions are stable between pH 2–10, precipitation can

occur below pH 2 solution and viscosity rapidly increase above pH 10 (Gopferich,

1997). Generally, solutions exhibit maximum viscosity and stability at pH 7–9.

Aqueous solutions may be sterilized by heating although this may results in some

reduction in viscosity. After autoclaving, viscosity is reduced by about 25% although

this reduction is less marked than for solutions prepared from material sterilized in

the dry state. Aqueous solutions stored for prolonged periods should contain an

antimicrobial preservative. The bulk material should be stored in a well-closed

container in a cool, dry, place (Parsons, 2006).

Viscosity-increasing agents are extensively used in the formulation of many

pharmaceuticals. In solution formulations, cellulose derivatives are used most

frequently. As increased viscosity can offer both advantages and disadvantages with

respect to use of the preparation, therefore, knowledge of viscosity and flow

properties is important in selecting appropriate ingredients. If sterility of the

preparation is required, the alteration in viscosity properties due to the sterilization

process and various additives, namely electrolytes, should be investigated

(Šklubalová and Zatloukal, 2011).

The viscosity modifying agents used in ophthalmology are water-soluble naturally

occurring, semi-synthetic or synthetic polymers (Shell, 1982; Lee and Robinson,

1986). The natural polymers include botanical polysaccharides (guar gum, locust

bean gum) semi-synthetic cellulose derivatives, such as cellulose ethers

29

(hydroxypropyl methylcellulose, hydroxyl propylcellulose, hydroxyl ethylcellulose,

methylcellulose and carboxy methylcellulose). Cellulose esters are

celluloseacetophtalate, microbial polysaccharides (dextran, xanthan gum, gellan,

gum and scleroglucan), algal polysaccharides (sodium alginate and carrageenan)

and animal polysaccharides (sodium hyaluronate, chondroitin sulphate and

chitosan).

Polyvinyl alcohol, polyvinylpyrrolidon and polyacrylic acid (Carbopol®) are commonly

used synthetic polymers (Sintzel et al., 1996). In general, viscous ophthalmic

solutions exhibit a pseudoplastic rheological behaviour. Pseudoplastic solutions offer

less resistance to movement of the eyelids over the globe and, therefore, are

expected to be more comfortable in the eye than Newtonian solutions (Dudinski et

al., 1983 and Van Ooteghem, 1987). The ideal viscosity of an ophthalmic solution is

estimated at 15–30 mPa·s, except in the case of viscoelastic polymers where a

higher viscosity is well tolerated by the patient (Ludwig & Van Ooteghem, 1988).

Ideally, small volumes of an ophthalmic solution should be instilled to diminish the

drainage rate and to reduce systemic absorption (Urtti & Salminen, 1993). To

produce eye drops with the ideal volume of 5–25 μl, dropper tips with small-

dimension capillaries are necessary. The inner aperture and outer orifice diameter

determine the flowing of the liquid through the capillary and, consequently, could

influence the viscosity and surface tension of the solution to be dispensed (Saettone

et al., 1984; Chang et al., 1988; Podder et al., 1992; Urtti and Salminen,

1993; Meseguer et al., 1996). In 1984, Jho and Carreras stated that the formation of

a drop under flowing conditions at the orifice of a calibrated capillary depended

primarily on the surface tension of the solution as according to Tate's law, but was

also influenced by hydrodynamic effects including drop formation rate, gravitational

and viscous forces.

In this study the following excipients were used as the viscosity modifying agent’s

carboxy methylcellulose sodium, glycerol and hydroxylpropyl methylcellulose (Rowe

et al., 2003). They were used to thicken and stabilize the formulation (Swarbrick et

al., 2000). They also lack reactivity with other excipients and PE and do not irritate

the eye (Ranucci & Silverstein, 1998).

30

2.4.2.5 Additives

Very few additives in eye drop solutions are permissible because they either reduce

the efficacy of the active pharmaceutical ingredient or are toxic for use in the ocular

region. Antioxidants like sodium bisulfite or sodium metabisulfite are permitted in

concentrations of up to 0.3%, particularly in solutions containing phenylephrine and

epinephrine salts. The antioxidant acts as a stabilizer to minimize oxidation of PE

and epinephrine. Other types of antioxidants are ascorbic acid and acetylcysteine

(Järvinen et al., 1995).

Non-ionic surfactants which are least toxic to the ophthalmic tissues are permitted at

concentrations of approximately 0.005%–0.2%. Surfactants are used as co-solvents

to increase solubility and to improve topical bioavailability of the API and to improve

the therapeutic response of an ophthalmic drug. Some surfactants can prolong

ocular residence time of the drug enhancing ocular permeation of the drug molecules

(Urtti & Salminen, 1993; Järvinen et al., 1995).

Benzalkonium chloride is a cationic surfactant and a preservative used frequently in

eye drop solutions. Concentrations that are used are in the range of 0.005–0.02%,

with toxicity the limiting factor on the concentration used. The benzalkonium cation

has a large molecular weight and is inactivated by macromolecules possessing an

opposite charge or by sorption. Benzalkonium chloride is the preservative of choice

and is used in many commercial eye drops, as it has low irritation potential to the

eye, has a broad-spectrum bactericidal activity at very low concentrations and is

compatible with many API (Winfield et al., 2009).

A common penetration enhancer is EDTA which loosens the tight junctions between

the superficial epithelial cells, facilitating paracellular transport (Saettone et al., 1996;

Hochman & Artursson, 1994). It is used in concentration of 0.1%.

The sorption characteristics of surfactants have to be identified before being included

in a formulation. The reason being is that surfactants can absorb preservatives and

thus render the formulation unpreserved (Baudouin et al., 2010).

31

Sodium metabisulfite has antioxidant, antimicrobial (in acidic preparations), and

antibrowning agent properties. In acidic preparations, sodium metabisulfite is used at

concentrations of 0.01–1% and less than 0.5% in alkaline preparations is slowly

oxidized to sodium sulfate on exposure to air and moisture. Sodium metabisulfite is

immediately converted to sodium ions (Na+) and bisulfite ions (HSO3+) when mixed

with water. The crystals also disintegrate when exposed to air and moisture.

Decomposition of aqueous sodium metabisulfite occurs as it exposed to air. The bulk

material should be stored in a well-closed container protected from light, in a cool,

dry, place (Stewart, 2006).

Edetic acid and edetate salts are used in ophthalmic pharmaceutical formulations as

chelating agents. Edetic acid and edetates are primarily used as antioxidant

synergists by sequestering trace amounts of metal ions, particularly copper, iron, and

manganese, which might otherwise catalyze autoxidation reactions. Edetic acid and

edetates may be used alone or in combination with true antioxidants, the usual

concentration employed being in the range 0.005–0.1% w/v. Edetic acid possesses

antimicrobial activity against Gram-negative microorganisms, Pseudonomas

aeruginosa, some yeasts, and fungi, although this activity is insufficient for edetic

acid to be used effectively as an antimicrobial preservative on its own. Many

solutions used for the cleaning, storage and wetting of contact lenses contain

disodium edentate. Edetic acid occurs as a white crystalline powder (Weller, 2006).

Aqueous solutions of edetic acid or edetate salt may be sterilized by autoclaving,

and should be stored in an alkali-free container. Edetic acid and edentates should be

stored in well-closed containers in a cool, dry, place (Weller, 2006).

The most widely used vehicle for pharmaceutical products which is also a solvent is

water. This is due to its physiological compatibility with many compounds and

absence of toxicity. It has a high dielectric constant which is necessary for ensuring

the dissolution of a wide range of ionisable materials (Mido et al., 2003).

There are two types of water used in the production of pharmaceutical products:

purified water and water for injections. Purified water must be free of ionic and

organic chemicals and microbial increase. It is produced by mains water going

through a unit that deionizes and distills the water, has an ion-exchange, provides for

32

reverse osmosis and finally filters. All purified water systems must be validated.

Purified water should be supplied constantly at ≥ 80 °C (Potdar, 2007).

Water for injection is used in the production of injections and other pharmaceutical

applications like cleaning of critical equipment and preparation of pharmaceutical

products. The water for injection gets its feed from purified water, which is subjected

to further distillation and reverse osmosis. The requirements for bacterial endotoxin

tests are much higher values as it must be free of microbial contamination and

endotoxins. Water for injection must show no reaction with the limulus amebocyte

lysate (LAL) reagent which is used to test for microbes (Potdar, 2007).

An important factor for eye drops is that they should be sterile when dispensed in a

multiple-application container (Reddy & Ganesan, 1996). Aseptic techniques during

the manufacture of injections have to be adopted when manufacturing sterile eye

drops (Turco & King, 2005).

Essentially two strategies are adopted in the manufacture of microbiologically

acceptable pharmaceutical formulations. The first and most important is to minimize

the introduction of microorganisms from sources such as air, water, raw materials

and personnel; and the second is to formulate the final product so as to be hostile to

microorganisms, by the addition of preservatives. Common waterborne organisms

found are the Pseudomonas-Achromobacter-Alcaligenes types, including

occasionally P. aeruginosa (McDonnell, 2007).

Purified water is a typical source of microorganisms as during use, the ion-exchange

column may become contaminated from the water passing through and the

entrapped organisms rapidly and multiplies to produce high counts in the outflowing

water. Water produced by reverse osmosis might present a problem if the osmosis

membrane is not disinfected at regular intervals. Distilled water may also be a

significant source of contamination, because the chlorine that protects tap water is

lost on distillation, and Gram-negative bacteria may grow to concentrations as high

as 105 to 106 ml-1 within a few days of storage at ambient temperature. With the

above in mind the correct approaches to preservation are based on two important

principles. The first is that a preservative must not be added to a product to mask

33

any deficiencies in the manufacturing procedures, and the second is that the

preservative should be an integral part of the formulation, chosen to afford protection

in that particular environment (Parker, 2002).

Sterility is one of the important characteristics for ophthalmic solutions. It is defined

as the absence of all living organism (McDonnell, 2007). This especially includes

microorganisms, such as bacteria, yeasts, molds, and viruses. The presence of even

one bacterium in an eye drop container renders its non-sterile. Sterility could be

determined or proved by inoculating the eye drop with microbiological cultural media.

If sterile, no microbial growth will be observed; if not, the culture medium will become

turbid as a result of microbial proliferation (Hanlon, 2002).

Serious ocular infection can result from the use of contaminated eye drop solutions.

Such solutions are the cause of corneal ulcers and loss of eyesight. Contaminated

solutions have been found in use and dispensed on prescription in community and

hospital pharmacies (Hodges, 2002).

Microbes commonly found as a contaminants are the Staphylococcus group.

Pseudomonas aeruginosa is a less frequent contaminant; however it is often found

as a contaminant in sodium flourescein. P aeruginosa is an opportunistic and very

dangerous organism that grows well on most culture media and produces both toxins

and antibacterial products. The latter tend to kill off other contaminants and allow

the P aeruginosa to grow in pure culture. This gram-negative bacillus also grows

readily in eye drop solutions, making it a source of extremely serious infections of the

cornea. Its rapid infection rate can cause complete loss of sight in 24 to 48 hours. In

concentrations tolerated by tissues of the eye, only certain antimicrobial agents are

effective against some strains of this organism (Hanlon, 2002).

Sterile eye drop solutions in multiple-dose containers can be contaminated easily.

For example, if a dropper bottle is used, the tip of the dropper while out of the bottle

can touch the surface of a table or shelf if laid down or it can touch the eyelid or

eyelash of the patient during administration. The solution may be an effective

antimicrobial agent, but the next use of the contaminated solution may occur before

enough time has elapsed for all of the organisms to be killed. In this manner living

34

organisms penetrate through an abrasion into the corneal stroma, within the corneal

stroma, traces of antimicrobial agents are neutralized by tissue components and the

organisms finds an excellent culture medium for rapid growth and dissemination

through the cornea and the anterior segment of the eye (Fassihi, 2001).

When the vitreous humour is infected by Bacillus subtils, it produces an abscess

which can cause blindness. Aspergillus fumigates is a pathogenic fungus found in

eye solutions and is responsible for accelerating deterioration of the active drugs. In

the late 1930s there was an epidemic of keratoconjunctivitis, it was caused by one

bottle of virus contaminated tetracaine solution. Virus contamination is difficult to

control because none of the preservatives available is virucidal. In addition, viruses

are not removable by filtration they are however destroyed by autoclaving. Recent

studies show that the adenoviruses (Type III and VIII), are causative agents of viral

conjunctivitis such as the epidemic keratoconjunctivitis (McDonnell, 2007).

2.5 Sterilization

Sterilization is defined as a process used to render a surface or product free from

viable organisms, including bacterial spores. Common methods of sterilization

include moist heat under pressure, dry heat, filtration, gas sterilization, and ionizing

radiation (McDonnell, 2007). A process, whereby a product is sterilized in its final

container and, which permits the measurement and evaluation of quantifiable

microbial lethality is called terminal sterilization. This process differs from aseptic

manufacture, which requires a series of sterile techniques and provides a passive

process of protection of the dosage form from contamination. If a finished product is

suitable for terminal sterilization, then this is the method of choice (Hanlon, 2002).

Sterilization is a requirement during the manufacture of eye products. The methods

used depend on the active ingredient, product resistance to heat and on the

packaging used. The sterile solution will usually contain an antimicrobial

preservative to protect against unintended contamination during use. Preservatives

are not used to produce a sterile but rather to maintain sterility (Prabhasawat et al.,

2005).

35

2.5.1 Steam under pressure as a method of sterilization

Terminal sterilization by autoclaving is an effective method of sterilization but the

solution components have to be heat-resistant in order to avoid degradation.

Effective sterilization is achieved when steam penetrates the autoclave load

uniformly. Products in their primary containers are autoclaved under pressure (1–2

atm) at 121 °C for 15 minutes. A technique has been developed which is called air-

over-steam autoclave. This allows pressure adjustments to be made during the

autoclave cycle to permit the autoclave sterilization of materials such as,

polypropylene containers that deform when exposed to heat (Lang et al., 2005).

2.5.2 Filtration as a method of sterilization

Filtration is defined as the process of removing solids or suspended matter in a liquid

or gas by passing matter through a porous medium where solids are retained or

entrapped (Twitchell, 2002). The USP (2004) states that sterile membrane filters

must have a pore size of 0.2 µm. Filtration is a method of sterilization as it offers the

substantial advantage of room temperature operation with none of the damaging

effects of exposure to heat or sterilizing gas. Filtration sterilization involves the

transfer of the finished sterile product into sterilized containers, through a filter paper

using aseptic techniques. The membrane filter is either hydrophobic or hydrophilic;

the right choice should be made based its compatibility with the API and excipients,

so as to reduce sorption. Sorption could significantly reduce the efficacy of the

manufactured product. The membrane filtration equipment itself usually is sterilised

by autoclaving. Filtration permits extemporaneous preparation of eye drop solutions

that have a high probability of being sterile under aseptic conditions (Gould, 2004).

2.5.3 Laminar-flow principles

A laminar-flow work area is used to prepare sterile, particulate free solutions.

Laminar-flow is defined as airflow in which the total body of air moves with uniform

velocity along parallel lines with a minimum of eddies. Laminar-flow with the HEPA

filters reduces the possibility of airborne microbial contamination by providing air free

of viable particles and free of practically all inert particulates. Laminar-flow units can

36

be found in different shapes and sizes and in two categories, horizontal and vertical

laminar-flow (Turco & King, 2005). Laminar-flow is not a guarantee of sterility and

correct procedures and sterile techniques are necessary (McHugh, 2010).

Laminar flow areas are supplied with air passed through high effiecincy particulate

filter (HEPA) filters which are high density mats composed of randomly arranged

fibres and are used as air filters to remove much smaller particles such as dust

pollutants and microbes. The fibres, made of fibreglass, have a diameter-range of

0.5 and 2.0 µm.

Factors that affect function of HEPA filters are fibre diameter, filter thickness, and

face velocity (Abdel-Magid & Caron, 2006).

The HEPA filters remove finer particles in air passed through them, and virtually free

it from foreign matter. Ultraviolet light (UV) is installed in the air ducts on the

downstream side of the filter to kill microbes that may have escaped through or

around the filter (Brooks et al., 2007). The hood air flow should have an air velocity

of 100 ft. / minute (Potdar, 2007).

2.5.4 Preservatives used in eye drop formulations

Preservative substances must be evaluated as part of the total eye drop preparation

in the proposed package. The growth or presence of microorganisms in the eye drop

can lead to destruction of the product or transmission of disease to the consumer.

Destruction can be in the form of chemical degradation of drugs or excipients by the

enzymes produced by the microorganism or a breakdown of the physical attributes

such as tactile change, visible change in colour, or smell (Amin et al., 2010).

The USP (2004) states that eye drop solutions may be packaged in multiple-dose

containers and must contain a substance or mixture of substances to prevent the

growth of, or to destroy, microorganisms introduced accidentally when the container

is opened during use. Eye drop solutions prepared and packaged for a single

application, need not contain a preservative because they are not intended for reuse.

http://en.wikipedia.org/wiki/Fiberglass

37

Preservatives are not to be used in solutions intended for intraocular use because of

the risk of irritation.

The selection of an eye drop preservative can be cumbersome because of the

relatively small number of suitable agents. As there is no ideal preservative,

however the following criteria are useful in preservative selection:

The agent should have a broad spectrum and be active against gram-positive

and gram-negative organisms as well as against fungi.

The agent should exert a rapid bactericidal activity, particularly against known

virulent organisms such as P aeruginosa strains.

The agent should be stable over a wide range of conditions for example

autoclaving temperatures and a wide pH range.

Compatibility should be established with other components in the product and

with packaging systems.

Lack of toxicity and irritation should be established with a margin of safety

(Lang et al., 2005).

Sterile eye drop solutions in multiple-dose containers can be contaminated easily.

For example, if a dropper bottle is used, the tip of the dropper, while out of the bottle,

can touch the surface of a table or shelf if laid down or it can touch the eyelid or

eyelash of the patient during administration (Amin et al., 2010). The solution may

have an effective antimicrobial, but the next use of the contaminated solution may

occur before enough time has elapsed for all of the organisms to be killed (Hodges,

2002). In this manner living organisms penetrate through an abrasion into the

corneal stroma, within the corneal, traces of antimicrobial agents are neutralized by

tissue components and the organism finds an excellent culture medium for rapid

growth and dissemination through the cornea and the anterior segment of the eye

(Fassihi, 2001).

The USP (2004) provides a test for preservative effectiveness; preservative

effectiveness as an immediate measure, its adequacy or stability as a function of

time must also be ascertained. This is achieved by measuring both chemical

stability and preservative effectiveness over a given period of time and under varying

38

storage conditions. Common compounds used as preservatives are discussed

below.

2.5.4.1 Quaternary ammonium compounds

Benzalkonium chloride is a quaternary ammonium compound and the most common

preservative used in eye drop formulations. Reviews have indicated that it is well

suited for use as an eye drop preservative. It has antimicrobial, antiseptic and

disinfectant properties (Kibbe, 2006). Benzalkonium chloride is found in 65% of

commercial eye drop products as a preservative (Rojanasakul et al., 1992). As a

cationic surface-active agent of high molecular weight it is not compatible with

anionic compounds, this defines its limitations, despite its broad use. It is

incompatible with salicylates and nitrates and inactivated by high-molecular-weight

non-ionic compounds (Le Bourlais et al., 1998).

Benzalkonium chloride has excellent chemical stability and desirable antimicrobial

characteristics. It is a mixture of alkyl benzyldimethylammonium chlorides ranging

from n-C8H17 through n-C16H33. The n-C12H25 homolog content is not less than 40%

on an anhydrous basis (Ali et al., 2009). It is one of the most widely used

preservatives, at a concentration of 0.01–0.02% w/v. It is used in combination with

other preservatives or excipients, particularly, 0.1% w/v disodium edetate, to

enhance its antimicrobial activity against strains of Pseudomonas (Rozet et al.,

2011).

Benzalkonium chloride occurs as a white or yellowish-white amorphous powder, a

thick gel, or gelatinous flakes. It is hygroscopic, soapy to the touch, and has a mild

aromatic odour and has a very bitter taste (Kibbe, 2006).

Benzalkonium chloride solutions are active against a wide range of bacteria, yeasts,

and fungi. Activity is more marked against Gram-positive than Gram-negative

bacteria and minimal against bacterial endospores and acid-fast bacteria. The

antimicrobial activity of benzalkonium chloride is significantly dependent upon the

alkyl composition of the homolog mixture. Benzalkonium chloride is ineffective

against some Pseudomonas aeruginosa strains, Mycobacterium tuberculosis,

39

Trichophyton interdigitale, and Trichophyton rubrum. However, combined with

disodium edetate (0.01–0.1%w/v), benzyl alcohol, phenylethanol, or phenylpropanol,

the activity against Pseudonomas aeruginosa is increased. Antimicrobial activity

may also be enhanced by the addition of phenylmercuric acetate, phenylmercuric

borate, chlorhexidine, cetrimide, or m-cresol. In the presence of citrate and

phosphate buffers (but not borate), activity against Pseudonomas can be reduced.

Benzalkonium chloride is relatively inactive against spores and moulds, but is active

against some viruses including human immunodeficiency virus (HIV). Inhibitory

activity increases with pH although antimicrobial activity occurs between pH 4–10

and organisms inhibited at specific concentrations can be seen below in Table 2

(Kibbe, 2006).

Table 2: Typical minimum inhibitor concentrations of benzalkonium chloride (Kibbe, 2006).

Microorganism MIC (µg/mL)

Aerobacter aerogenes 64

Clostridium histolyticum 5

Escherichia coli 16

Pseudomonas aeruginosa 30

Salmonella typhosa 4

Staphylococcus aureus 1.25

Staphylococcus pyrogenes 1.25

Vibrio cholera 2

Benzalkonium chloride is hygroscopic and may be affected by light, air, and metals.

Solutions are stable over a wide pH and temperature range and may be sterilized by

autoclaving without loss of effectiveness. Solutions may be stored for prolonged

periods at room temperature. It is incompatible with aluminium, anionic surfactants,

citrates, flourescein, hydrogen peroxide, iodides, kaolin, lanolin, nitrates, and non-

ionic surfactants in high concentration (Kibbe, 2006).

Other types of quaternary ammonium compounds occasionally used include

benzethonium chloride and cetyl pyridinuim chloride. Few quaternary ammonium

compounds have been formulated, by attaching soluble high molecular weight

polymers (Ali et al., 2009).

40

2.5.4.2 Parahydroxybenzoic acid esters

Mixtures of methylparaben and propylparaben are sometimes used as ophthalmic

antimicrobial preservatives. The concentration of methylparaben used is in the range

of 0.1 to 0.2%, while that of propylparaben approaches its solubility in water (

0.04%). They are not considered efficient bacteriostatic agents and are slow in their

antimicrobial action. Ocular irritation and stinging are attributed to their use in eye

drop preparation (Furrer et al., 2002).

Propylparaben is widely used as an antimicrobial preservative in pharmaceutical

formulations. It may be used alone, in combination with other paraben esters, or

with other antimicrobial agents. It is effective over a wide pH range and has a broad

spectrum of antimicrobial activity although it is most effective against yeasts and

moulds. It is known for its poor solubility; particularly the sodium salt. This may cause

the pH of poorly buffered formulations to become more alkaline. Use in ophthalmic

formulations is in a concentration range of 0.005–0.01%, Table 3 shows organisms

inhibited at various concentrations. Propylparaben is found as a white, crystalline,

odourless, and tasteless powder (Rieger, 2006b).

Table 3: Minimum inhibitory concentration for propylparaben in aqueous solution (Rieger, 2006b)


Aspergillus niger ATCC 9642 500

Bacillus subtilis ATCC 6633 500

Candida albicans ATCC 10231 250

Escherichia coli ATCC 8739 500

Klebsiella pneumonia ATCC 8308 500

Pseudomonas aeruginosa ATCC 9027 >1000

Salmonella typhosa ATCC 6539 500

Staphylococcus aureus ATCC 6538P 500

Staphylococcus epidermidis ATCC 12228 500

Trichophyton mentagrophytes 65

Aqueous propylparaben solutions at pH 3–6 can be sterilized by autoclaving, without

decomposition. At pH 3–6 aqueous solutions are stable for up to about 4 years at

41

room temperature while solutions at pH 8 or above are subject to rapid hydrolysis

(Rieger, 2006b).

Methylparaben may be used either alone, in combination with other parabens, or

with other antimicrobial agents. Preservative efficacy is also improved by the addition

of 2–5% propylene glycol, or by using parabens in combination with other

antimicrobial agents such as imidurea (Rieger, 2006a). In ophthalmic formulations it

is used between 0.015–0.2 percent. Methylparaben occurs as colourless crystals or

a white crystalline powder. It is odourless or almost odourless and has a slight

burning taste (Rieger, 2006a).

Methylparaben exhibits antimicrobial activity between pH 4–8. Preservative efficacy

decreases with increasing pH due to the formation of the phenolate anion. Parabens

are more active against yeasts and moulds than against bacteria, Tables 3 and 4

show ophthalmic concentrations and organisms inhibited respectively. They are also

more active against Gram-positive bacteria than against Gram-negative bacteria.

Aqueous solutions of methylparaben, at pH 3–6, may be sterilized by autoclaving at

120 ˚C for 20 minutes, without decomposition. Aqueous solutions at pH 3–6 are

stable for up to about 4 years at room temperature, while aqueous solutions at pH 8

or above are subject to rapid hydrolysis (Rieger, 2006a).

Table 4: Minimum inhibitory concentrations of methylparaben in aqueous solution (Rieger, 2006a).


Aspergillus niger ATCC 10254 1000

Bacillus subtilis ATCC 6633 2000

Candida albicans ATCC 10231 2000

Escherichia coli ATCC 8739 1000

Klebsiella pneumonia ATCC 8308 1000

Pseudomonas aeruginosa ATCC 9027 4000

Salmonella typhosa ATCC 6539 1000

Staphylococcus aureus ATCC 6538P 2000

Staphylococcus epidermidis ATCC 12228 2000

Trichophyton mentagrophytes 250

42

2.6 Efficacy of antimicrobial preservation

Pharmaceutical formulations that do not have adequate antimicrobial activity are

protected with antimicrobial preservatives particularly in aqueous formulations. This

is to prevent the proliferation or to limit microbial contamination which, during normal

conditions of storage and use, particularly for multidose containers, could occur and

present a hazard to the patient. The efficacy of an antimicrobial preservative may be

enhanced or diminished by the active constituent of the preparation or by the

formulation in which it is incorporated or by the container and closure used. The

antimicrobial activity of the formulation in its final container is investigated to ensure

that its activity has not been impaired by storage. Such investigation may be carried

out on samples removed from the final container immediately prior to testing (B.P,

2011).

During development of a pharmaceutical formulation, it should be demonstrated that

the antimicrobial activity in the formulation including preservatives provides adequate

protection from adverse effects that may arise from microbial contamination or

proliferation during storage and use of the preparation. The efficacy of the

preservatives on microbial activity can be achieved by tests which consist of

challenging the preparation in its final container, with prescribed inoculums of

suitable micro-organism, storing the inoculated formulation at a prescribed

temperature, withdrawing samples from the container at specified intervals of time

and counting the organisms in the samples removed (B.P, 2011).

The preservative properties of the formulation are adequate if, in the conditions of

test, there is a significant fall or no increase, as appropriate, in the number of micro-

organisms in the inoculated product after the times and at the temperatures

prescribed (B.P, 2011).

Laboratories can isolate different strains of desired micro-organisms by swabbing

infected patients, isolating strains from contaminated food, cosmetic or

pharmaceutical products and many other sources. Therefore strains obtained in

various manners vary in resistance to antimicrobial chemicals. Strains obtained from

human and animals are more resistant to antimicrobial chemicals than those from

43

other sources. Strains derived from contaminated medicines will be more resistant to

preservatives. This is because the characteristics of the organism (including its

resistance to antimicrobial chemicals) over a period of time changes as a result of

mutation and natural selection (Curtin and Cormican, 2003). To get results that are

reproducible by a variety of laboratories it is necessary to specify the strain of the

organism used for the experiment. The common strains used are cultures of

Escherichia coli, Candida albicans, Pseudomonas aeruginosa, Staphylococcus

aureus. Methods used to assess the activity of antimicrobial preservatives involve an

inoculum of the test organisms which are added to a solution of the product in

question. Samples are then removed over a period of time, the preservative is

inactivated and the surviving cells calculated (Brooks et al., 2007).

In all cases of measurement of antimicrobial activity it is necessary to standardise

and control factors such as the concentration of the test organism, its origin (species

and strain employed), the culture medium in which the cells are placed, temperature

and the of incubation time of cells after exposure to the chemical (Brooks et al.,

2007).

2.7 Packaging

According to the American Society for Testing and Materials, a plastic is a material

that contains, as an essential ingredient, one or more polymeric organic substances

of large molecular weight, is solid in its finished state and at some stage of

manufacture or processing into finished articles it can be shaped by flow (Rabinow

& Roseman, 2005). Important mechanical properties required in plastic packaging

materials are: tensile strength, impact strength, stiffness, flex resistance, tear

strength, coefficient of friction, blocking, fatigue resistance and creep failure

(Massey, 2004).

Optical properties are important qualities needed in the plastics as a method of

packaging. Some needed characteristics are light transmission, clarity, haze and

gloss (Marriot, 2006).

44

Ophthalmic glass containers with glass droppers have largely been replaced by

plastic bottles, only in rare cases are glass containers still in use (Van Santvliet &

Ludwig, 2004). Plastic containers are used more often due to their increased

resistance to shock, they are lightweight, and they present more options with regard

to design opportunities (Jenkins & Osborn, 1993).

Plastic containers used to store ophthalmic products should have the following

characteristics:

Flexibility (ability to deform).

Elasticity (ability to return to its original form after deformation).

Stiffness (resistance to deformation) (Smith and Hui, 2004).

Most plastic containers are shaped with round or oval bases containing substances

of 3 to 15 ml (Santvliet & Ludwig, 2004). There are disadvantages in plastic

containers, these are:

Minimal transparency (poor visuals of solutions inside the containers).

Low-density polyethylene allows permeability to vapours and light.

Adsorption of molecules from its contents and cracking due to intensive use

(McDonald and Parkin, 1995).

Plastic packaging is not interchangeable with glass (Shah et al., 2008). Plastic

packages may contain a variety of extraneous substances such as mould-release

agents, antioxidants, and reaction quenchers amongst others which can readily

leach out of the plastic and into the contained solution (Tokiwa & Calabia, 2004) and

label glues, inks and dyes also have been reported to penetrate (Van Santvliet &

Ludwig, 2004). Commercially-produced plastic eye drop bottles are made of

polyethylene or polypropylene. They are common containers for various drugs

including phenylephrine hydrochloride solutions (Winfield et al., 2009).

2.9 Formulation development

Each formulation and API is unique. Formulation experiments begin with a well-

structured formulation plan. The formulation strategy is a result of thorough analysis

45

of the preformulation data report and manufacturing process. The following criteria

are to be met in order to begin formulation development:

Relevant patents have been accessed and investigated.

The appropriate literature search has been sourced.

The regulatory and formulation strategies have been well-known.

The desired APIs have been ordered and received (Al-Shaalan, 2010).

A beneficial approach to formulation development of a generic product is to critically

evaluate and where possible, to characterize, the innovator product with respect to

composition and other qualitative and or quantitative analyses which may be

feasible. A simple approach to achieve the above analysis, is to determine the pH of

the innovator drug product dispersed in a small volume of pH adjusted purified water,

and then to compare the result with that yielded by a similar dispersion of the trail

formulation. The approach is based on the principle that if two dispersions provide

comparable pHs’, the excipients compositions of the innovator and generic

formulations are probably similar. However caution is needed since this test may not

be sufficient to provide all results needed. Initial trials and selection of batches

should be done using similar excipients. Possible instability or incompatibility can be

overlooked, as long as the same excipients provided in text are used. Small changes

in the concentration of key excipients can alter the appearance and physical

attributes, while impact on the drug product stability and dissolution can be

significant (Miller & Ermer, 2005).

The assessment of the final formulation can be achieved by scaling to require pilot

batches, and packaged into all possible configurations intended for future

commercialization. The batch is placed into accelerated (40 °C/75%RH) storage

conditions for a period of three months. The batches are tested using validated

analytical methods, should the batch prove to be stable over the 3 months period of

exposure, it would have a high degree of probability that the product is stable and

formulae succeeded. It is pivotal to ensure that all desirable characteristics are

achieved. The generic drug product must demonstrate the minimum 3 months

satisfactory stability, before the following are achieved:

46

Development of specifications for both API and the dosage form.

Ordering of the API and excipients for batch manufacturing.

Ordering of all relevant tooling, change parts.

Completion of development report (Ansel et al., 2011).

2.9.1 Validation of HPLC analytical methods

Guidelines provided by the international conference on harmonization are deemed

paramount for the registration of pharmaceuticals for human use as they represent

the regulatory agencies of the three largest pharmaceutical markets (U.S. Food and

drug administration, European Commission for the European Union, and Japanese

ministry for Health and Welfare (MHW)). The three regulatory agencies came

together to address issues of efficacy, safety, quality and harmonized guidelines.

Validation is a basic requirement to ensure quality and reliability of the results for all

analytical applications (Miller & Ermer, 2005). It is a requirement by registration

bodies like the MCC and FDA that the method be validated. A typical validation

technique would be the use of HPLC for its pharmaceutical analysis (Karcher et al.,

2005). Physical separation technique employed by HPLC is conducted in the liquid

phase in which a sample is separated into its constituent components (or analytes)

by distributing between the mobile phase (a flowing liquid) and a stationary phase

(sorbents packed inside a column). An online detector used by the HPLC monitors

the concentration of each separated component in the column effluent and

generates a chromatogram (Cecil & Sheinin, 2005). These detectors are then able to

quantify the major components in a purified sample, differentiate among components

of a reaction mixture and show trace impurities in a complex sample matrix (Wells,

2002).

Reversed-phase HPLC is the method of choice for stability-indicating assays

because the samples are generated in aqueous solutions. The advantages of

reversed-phase chromatography are due to convenience, a broad range of samples

that can be chromatographed conveniently and the speed of column equilibration is

shorter when compared to normal–phase chromatography (Nakashima et al., 2002;

Marin et al., 2005; Wong et al., 2006; Palabiyik & Onur, 2007; Heydari, 2008; Das

47

Gupta & Parasrampuria, 1987; Bastos & de Oliveira, 2009). Most reversed phase

HPLC separations are done in the isocratic mode, where the composition of the

mobile phase is held constant during the analysis. The external standard method is

the most general method for determination of the concentration of a compound in an

unknown sample. It involves the construction of a calibration plot using external

standards of the analyte. A fixed volume of each standard solution of known

concentration is injected onto the column and the peak response of each injection is

plotted versus the concentration of the standard solution. The standards are called

‘external standards’ because they are prepared and analyzed separately from the

unknown samples. After constructing the calibration plot, the unknown sample is

prepared, injected and analyzed in exactly the same manner. The concentration of

the analyte in the unknown sample is then determined from the calibration plot

(Wong, 2006).

Other types of standards are the internal standard, mass balance and area

normalization. The internal standard procedure allows the analyst to identify a region

of the chromatogram that is devoid of peaks (quiet region). The analyst then

attempts to identify a compound of known purity, that is structurally related to the

analyte, and which elutes in the quiet region of the chromatogram. The internal

standard should have a relative response factor that is about the same as the

analyte (Cecil & Sheinin, 2005).

Reversed phase HPLC has been extensively used for the pharmaceutical analysis of

phenylephrine hydrochloride in pharmaceutical dosage forms (Sprieck, 1974;

Muhammad and Bodnar, 1980; Chien & Schoenwald, 1985; Zeise et al., 1996;

Muszalska et al., 2000; Goicoechea & Olivieri, 2001; Savic et al., 2008; Samadi–

Maybodi & Nejad–Darzi, 2009; & Al-Shaalan, 2010).

2.9.1.1 Stability indicating HPLC analysis

Analytical procedures used for the assay of the API alone or in the final product

during stability studies should be stability indicating. A stability indicating assay is

one that accurately quantifies the API without interference from degradation

products, process impurities, excipients, or other potential impurities. Samples are

48

obtained by placing the pure API under stress intentionally, for example, by

subjecting the API to acid, base, heat, light or oxidation. This process is often called

forceful degradation or purposeful degradation. Usually, the goal is to degrade the

parent API by 10–20%. Degradation much greater than 10–20% could result in

secondary degradants that will complicate the development process (ICH

Harmonised Tripartite Guideline Q2A, 1994).

In the mass balance approach, all impurities are quantified and subtracted from the

absolute API value of 100%. This approach will result in a purity value that, if all

impurities are accounted for, is more accurate than the external or internal standard

methods. However, the ability to identify all impurities in a given drug substance may

require the use of hyphenated detection techniques and could be extremely costly to

complete on a regular basis. A related approach is called area normalization, and is

often used where the majority of the impurities can be identified and quantified in a

single chromatogram. All of the impurities would be assumed to have the same

relative response factor as the parent drug. The quantification of the individual

impurities would be reported as a percentage of the parent drug rather than an

absolute value in milligrams (Cecil & Sheinin, 2005).

2.9.1.2 Choice of analytical column and conditions

The C18 (2)-bonded phase [-CH2-(CH2)16-CH3] is the separation material contained

within columns used in the early development of HPLC. It is available in a wide

variety of forms and differs based on for example, silica type, pore size, surface

area. As most laboratories with HPLC equipment will have a C18 column available, it

is the first choice for initial experiments. In addition, with the broad range of

applications accessible in the literature or from commercial sources, it is often easy

to find a separation that is similar, allowing for selection of mobile phase conditions

that are likely to be suitable for solving a particular analytical problem. C18 columns

were utilised most prevalently in the analytical HPLC methods sourced in the

literature for the quantitative determination of phenylephrine hydrochloride solutions.

The advantages noted for the C18 (2)-bonded are flexibility in pH, ligand stability at

acidic pH and hydrolytic stability of the bonded phase based on a bi silane at low pH

49

which is five times higher than that of a monofunctional bonded phase (Saettone et

al., 1980; O’Donnell & Gillibrand, 1995; Palabiyik & Onur, 2007).

An ion-pairing agent is commonly used with reversed–phase chromatography as it is

a better alternative to ion–exchange chromatography for the separation of ionic

species. The most common ion–pairing agents are the tetrabutylammonium salts

and alkylsulphonic salts (octane-1-sulfonic acid sodium salt). Properties of the two

are as follows:

Tetrabutylammonium salts are:

Buffered to pH 7.5.

Form ion pairs with strong and weak acids.

Suppresses weak base ions.

While alkylsulphonic salts are:

Buffered to pH 3.5.

Form ion–pairs with strong and weak bases.

Buffering suppresses weak acid ions.

The longer the alkyl chain, the greater is the retention time (Florence and

Attwood, 2006).

Ion–pairing agents and pH increase the retention time with increasing concentration

of ion–pairing agent. The true origins of the ions which pair with drug ions is not

clear, but there is evidence that ion–pair formation aids absorption. Ion–pairing

provides the interaction between a drug ion and an organic ion of opposite charges

to form an absorbable neutral species. The formation of ion pairs will depend on

solvent–ion interactions: hydrophobic ions might be encouraged to form ion pairs by

the mechanism of water-structure enforced ion–pairing in which water attempts to

minimize the disturbance on its structuring, and achieves this end by reducing the

polarity of the species in solution by ion–pair formation (Florence and Attwood,

2006).

50

2.9.1.3 Steps for HPLC method validation

Method validation is the process of demonstrating that analytical procedures are

suitable for their intended use and that they support the identity, strength, quality,

purity and potency of pharmaceutical substances and products. The process of

validating a method cannot be separated from the actual development of the method

conditions, because the developer will not know whether the method conditions are

acceptable until validation studies are performed. Results of validation studies may

indicate that a change in the procedure is necessary, which may then require

revalidation. During each validation study, key method parameters are determined

and then used for all subsequent validation steps. The method developed for this

study was validated according to the International Conference on Harmonization

guidelines and the following were determined for the HPLC method:

Specificity.

System suitability.

Linearity.

Accuracy.

Precision.

Limit of detection.

Limit of quantification.

Range (ICH Harmonized Tripartite Guideline Q2A, 1994).

2.9.1.4 Linearity

Linearity of an analytical procedure is its ability, within a given range, to obtain test

results that are directly proportional to the concentration of analyte in the sample

(ICH Harmonized Tripartite Guideline Q2A, 1994). The requirements for linearity are

that the correlation coefficient of the regression line must be greater than or equal to

0.9999 and that the y-intercept must not be significantly different from zero (ICH

Harmonized Tripartite Guideline Q2A, 1994).

51

2.9.1.5 Accuracy and precision

Accuracy of an analytical procedure expresses the closeness of agreement between

the value that is found and the value that is accepted (either as a conventional true

value or an accepted reference value). Precision of an analytical procedure

expresses the closeness of agreement (degree of scatter) between a series of

measurements obtained from multiple sampling of the same homogenous sample

under the prescribed conditions (ICH Harmonized Tripartite Guideline Q2A, 1994).

The requirement for accuracy is that the percentage recovery of API for each

solution prepared must be within the 98.00 to 102.00% limit. The requirement for

precision is that the relative standard deviations at any one concentration must be

less than or equal to 2.00%.

2.9.1.6 Limit of detection and limit of quantification

The limit of detection (LOD) is the lowest analyte concentration that produces a

response detectable above the noise level of the system and the limit of

quantification (LOQ) is the lowest level of analyte that can be accurately and

precisely measured (Kupiec et al., 2004). The LOD is thus the lowest concentration

for which the relative standard deviation of multiple injections is less than 5.0%. By

convention, the LOD value is taken as 0.3 times the LOQ (Thomsen et al., 2003).

2.9.1.7 Range

Range of an analytical procedure is the interval between the upper and lower

concentrations of analyte in the sample (including these concentrations) for which it

has been demonstrated that the analytical procedure has a suitable level of linearity,

accuracy and precision (ICH Harmonized Tripartite Guideline Q2A, 1994).

2.9.1.8 Specificity

Specificity of an analytical procedure is its ability to assess, unequivocally, the

analyte in the presence of components that may be expected to be present (ICH

Harmonized Tripartite Guideline Q2A, 1994). Specificity suggests that a given

52

analyte peak is indeed the desired analyte structure and is 100% pure (Krull &

Swartz, 2001). The peak purity results from the photodiode-array analysis must show

that the phenylephrine hydrochloride peak is pure. In other words the purity angle

must be less than the threshold angle. Depending on the wavelength, a tungsten

lamp and a deuterium lamp are used as light sources. The polychromatic light beam

is focused on a flow-cell and subsequently dispersed by a holographic grating or

quartz prism. The spectral light then reaches a chip that contains 100 to 1000 light-

sensitive diodes arranged side by side. Each diode only registers a well-defined

fraction of the information and in this way all wavelengths are measured at the same

time. At the end of the run, a three-dimensional spectrochromatogram (absorbance

as a function of wavelength and time) is stored on the computer and can be

evaluated qualitatively and quantitatively. Peak identity and peak homogeneity (peak

purity) can be investigated by analyzing spectrum collected during chromatographic

seperations and detection, a pure compound will produce a peak with spectra that

have the same shape across the peak. In contrast any interefence from coeluting

analytes will produce composite spectra with varying degrees of spectra

dissimilarities across the peak. This is the basis of peak purity index. Diode array

detection offers the advantage that knowledge of the spectra of compounds of

interest enables interfering peaks to be eliminated such that an accurate

quantification of peaks of interest can be achieved despite less than optimal

resolution. Peak purity is of the utmost importance in the quality control of

pharmaceutical products (Kupiec et al., 2004).

Figure 4: Diagram of a typical HPLC-UV absorbance peak and plots of noise (or threshold) and purity angles (Krull & Swartz, 2001).

Figure 4 above shows a typical HPLC-UV absorbance peak with plots of noise and

purity angles. Stress testing is undertaken to demonstrate specificity when

53

developing stability indicating methods (Reynolds et al., 2002). The API and finished

products will be subjected to stress studies in order to force phenylephrine

hydrochloride degradation and thereby verify or exclude the presence of co-eluting

impurities or degradation products in the mobile phase, solvent or unstressed /

stressed solutions.

Impurities should be between 10 – 20 % of the API. This should include samples

stored under relevant stress conditions such as: light, heat, humidity, acid/base

hydrolysis and oxidation. Peak purity tests are used to show that the analyte’s

chromatographic peak is not attributable to more than one component (ICH

Harmonized Tripartite Guideline Q2 (R1), 2005).

2.9.2 Active-excipient compatibility studies

Impurities can be formed in pharmaceutical products as a result of an interaction

between the active ingredient and excipient introduced by formulation. In an ideal

product, the excipient used in the formulations should not interact with the drug

substance or introduce unwanted substances capable of accelerating the formation

of new impurities. Incompatibility found between drugs and excipients in

pharmaceutical products can alter the stability and bioavailability of drugs, thereby

affecting its safety and efficacy. Stress studies of the active-excipients mixture

generate high amounts of degradation products which can be identified using various

analytical procedures even though the potential degradation products are in low

concentrations (Douša et al., 2011).

Binary mixture compatibility testing is a commonly used method. In this approach,

binary (1:1 or customized) mixtures of the drug and excipient, with or without, added

water and sometimes compacted or prepared as slurries, are stored under stressed

conditions (also known as isothermal stress testing (IST)) and analyzed using a

stability-indicating method, e.g. HPLC. The water slurry approach allows the pH of

the drug-excipient blend and the role of moisture to be investigated. Alternatively,

binary mixtures can be screened using other thermal methods, such as differential

scanning calorimetry (DSC) (Clas et al., 1999).

54

DSC is a technique that is used extensively in the field of pharmaceutics. The main

benefit of DSC, compared to stress storage methods, is its ability to quickly screen

potential excipients for incompatibilities derived from the appearance, shifts or

disappearances of peaks and/or variations in the corresponding peak. DSC is mostly

used to show instability resulting from solid-solid interactions. Another feature of

DSC possesses is low sample consumption making it an attractive method. Although

DSC is unquestionably a valuable technique, interpretation of the data may not be

straightforward. In this method, the sample is exposed to high temperatures (up to

300 °C or more), which in reality is not experienced by the dosage form. Thus, DSC

results should be interpreted carefully, as the conclusions based on DSC results

alone can be often misleading and inconclusive. The results obtained with DSC

should therefore always be confirmed with isothermal stress testing, IST (Giron,

1998).

Isothermal stress testing involves storage of drug-excipient blends with, or without

moisture, at elevated temperatures for a specific period of time, typically 3–4 weeks

to accelerate drug degradation and interaction with excipients. The samples are then

visually observed for any changes in color or physical characteristics, and the drug

content, along with any degradants, is determined quantitatively. Although more

useful, the disadvantage with this method is that it is time consuming and requires

quantitative analysis using e.g. HPLC (Verma & Garg, 2004).

Ideally, both techniques, DSC and IST should be used in combination for the

selection of excipients. That was not the case in this research study and only IST

was used as it could satisfactorily indicate the results.

2.10 Determining formulation stability study

Stability, with respect to drug dosage form, refers to the chemical and physical

integrity of the dosage unit and the ability of the dosage unit to maintain protection

against microbiological contamination. The shelf life of the dosage form is the time

lapse from initial preparation to the specified expiration date. The product within its

shelf life must fulfil the monograph specification for identity, strength, quality, and

55

purity. Stability parameters of a drug dosage form can be influenced by

environmental conditions of storage (temperature, light, air, and humidity) as well as

packaging (USP, 2004).

Stability studies on dosage forms should be conducted by means of specific

temperatures and relative humidities representing storage conditions experienced in

the distribution chain of climatic zones of South Africa (climatic zones I and II).

Stability studies performed on eye drops in the pharmaceutical industry follow an

integral part of a formulation development programme. The standardized approach

applied to the formulation or design of the eye drop is similar to those applied to

other dosage forms, which are:

Selection of batches.

Test procedure and criteria.

Specification storage test conditions.

Testing frequency.

Packaging material.

Evaluation statements and labelling (Miller–Meeks et al., 1991).

Stability study is one of the most important areas, in relation to the registration of

pharmaceutical products, as it predicts shelf life and storage instructions for batches.

It also determines degradation of products, mechanism of breakdown and conditions

under which the breakdown occurs. With the help of stability studies, any parameter

subject to change within the eye drop during storage can be measured, such as

appearance, pH, viscosity and density, (where relevant), solubility time

(reconstitution and appearance thereof) sterility, preserving ability and preservative

content (where relevant). Tests are also performed to ensure compatibility between

the container-closure system and the product. Stability testing is the cornerstone of

drug development or formulation (Jeffs, 2009).

During storage, one or more of the following changes may take place:

a) Chemical interaction involving drug, excipients or container, or many

combinations of these.

b) Alteration of physical form.

56

c) Mould or bacterial growth commonly referred to as biological change.

Their effect may be to make the preparation unusable on account of:

i. Loss of active agent.

ii. Development of a toxic decomposition product.

iii. Poor aesthetics to the patient.

Chemical change involves the drug itself because this is normally the most reactive

component of the system. Occasionally, it doesn’t involve the drug but is limited to

the excipient and/or the container for example the rusting of cans or flaking of glass,

this thus gives plastics added advantage. A hazardous occurrence within plastic is

the extraction of substances from the walls of plastic containers. Other chemical

changes, such as discoloration, may be harmless and quantitatively negligible but

could have serious effect on the acceptability of the product. Chemical change

occurs when stimulated by heat light, moisture and aeration. Thus antioxidants,

buffers and chelating agents are commonly added to offer protection against light

and moisture. Physical change may make a product inconvenient or impossible to

use and occasionally can lead to danger to patient. The growth of micro-organisms

can cause spoilage either by their appearance or because they have induced

significant chemical change (Huynh-Ba, 2009).

57

3. METHODOLOGY

3.1 HPLC method validation

3.1.1 Equipment

The HPLC system consisted of a complete FPLC Shimadzu® HPLC system which

has a SPD-M20A Prominence diode array detector, SIL-20A Prominence auto-

sampler, DGU-20A5 Prominence degasser, LC-20AB Prominence liquid

chromatography and CTO-10AS vp Prominence column oven ( Shimadzu, Tokyo,

Japan). The stationary phase consisted of a reverse phase Phenomenex® Luna C18

(2) column 250 mm × 4.60 mm, 5 μm particle size (Separations, Johannesburg, SA).

3.1.2 Materials and reagents

Phenylephrine hydrochloride, sodium citrate dihydrate, boric acid, disodium ededate,

sodium metabisulphite, and benzalkonium chloride were kindly donated by Aspen

Pharmacare (Port Elizabeth, SA). Water for chromatography was produced by an

Ultra Clear TWF/El-Ion® system which was pre-treated and made ultrapure (reverse

osmosis) (Separations, Johannesburg, SA). HPLC grade methanol and octane-1-

sulfonic acid sodium salt were obtained from Sigma-Aldrich (Pty) Ltd (Kempton Park,

SA). Analytical/technical grades of sodium hydroxide pellets, carboxy

methylcellulose sodium, hydroxylpropyl methylcellulose, glycerol, hydrochloride acid

solution 33% and pyrophosphoric acid (phosphoric acid) were obtained from Merck

Laboratory Supplies (Pty) Ltd (Midrand, SA) and hydrogen peroxide 30% was

sourced from Saarchem (Pty) Ltd (Johannesburg, SA).

3.1.3 Mobile phase preparation and standard curve construction

A mass of 1.1 g of octane-1-sulfonic acid sodium salt was dissolved in a Schott

bottle that contained one litre of a mixture of methanol and water (1:1). The pH was

adjusted to 3.0 with pyrophosphoric acid. The resulting solution was mixed,

degassed by ultrasonication (Ultrasonic LC 130, Labotec, Germany) and vacuum

filtered through a 0.45 μm Millipore filter (Millipore Corporation, Bedford,

58

Massachusetts, USA) prior to use. The dilution solvent was prepared by mixing

HPLC grade methanol and water in a 1:1 ratio and adjusted to a pH of 3 with

pyrophosphoric acid.

The standard curve: Phenylephrine hydrochloride stock solution was prepared by

accurately weighing 2 mg of phenylephrine hydrochloride material into a 100 ml

volumetric flask, dissolving it in dilution solvent and making up to volume with dilution

solvent. Calibration standards containing 0.0125, 0.025, 0.05, 0.075, 0.1 and 0.15

mg/ml were prepared by making appropriate solvent dilutions of the working stock

solution. Five millimetre of calibration standard was filtered through a 0.45 μm

Millex® Syringe Driven Filter Unit prior to injection

3.1.4 Chromatographic conditions

The flow rate was set at one millimetre per minute. The column temperature was set

at its temperature of 40 °C and column back-pressure varied between 2800 to 3000

psi. A PDA was used, therefore all runs were analysed at wave length, 280 nm. The

injection volume was 20 μl (USP, 2004). The assay was performed utilizing a reverse

phase Phenomenex® Luna C18 (2) column (250 mm × 4.60 mm, 5 μm particle size).

To identify the phenylephrine hydrochloride, the retention times of the peaks were

noted and to quantitate the amount of the API, values found of AUC (peak area)

which is computer-stored and generated was used. A graph of the concentrations vs.

peak area was plotted. Equation of the line was calculated using Microsoft Excel®

program. With the above, the following were determined: specificity, system

suitability, linearity, accuracy, precision, limit of detection/limit of quantification and

range.

3.1.5 Linearity

A 2 mg/ml stock solution was prepared and diluted to concentrations of 0.0125,

0.025, 0.05, 0.075, 0.1 and 0.15 mg/ml using the dilution solvent. The concentration

of 0.1 mg/ml was defined as 100% while 0.15 mg/ml was taken to be 150%. Each of

the concentrations was assayed in triplicate. The calibration curve was constructed

59

by plotting the peak areas of phenylephrine hydrochloride versus the respective

phenylephrine hydrochloride concentrations and a linear regression trend line was

fitted to the plot on Microsoft Excel® 2007, (Microsoft Corporation).

3.1.6 Accuracy and precision

Accuracy and precision were determined by replicate injection (n=3 and 6,

respectively) of three phenylephrine hydrochloride solutions, at the upper, middle,

and lower limits of the concentration range studied. The accuracy of a measurement

system is the degree of closeness of measurements of a quantity to that quantity's

actual (true) value (Green, 1996). The precision of a measurement system, also

called reproducibility or repeatability, is the degree to which repeated measurements

under unchanged conditions show the same results (ICH Harmonized Tripartite

Guideline Q2A, 1994). The concentration ranges were 0.0095 mg/ml (lower limit),

0.054 mg/ml (middle limit) and 0.138 mg/ml (upper limit). The theoretical

concentrations were calculated from the linear regression curve, and compared to

the actual concentrations tested. The actual mean concentration and standard

deviation were calculated at each theoretical concentration. The mean

concentrations and percentage recovery of phenylephrine hydrochloride obtained for

the replicate injections were a measure of the accuracy of the method, whilst the

relative standard deviations at any one concentration provided a measure of

precision. The requirement for accuracy is that the percentage recovery of

phenylephrine hydrochloride for each solution prepared must be within the 98.00 to

102.00% limit. The requirement for precision is that the relative standard deviations

at any one concentration must be less than or equal to 2.00%.

3.1.7 Limit of detection and limit of quantification

Sensitivity of the method was determined by means of the detection limit (LOD) and

quantification limit (LOQ). Calculations for LOD were based on the standard

deviation of the calibration curve (σ) and the slope of curve (S), using the equation

LOD = 3.3 × σ divided by S. LOQ was calculated using the equation LOQ = 3.33

multiplied by LOD (ICH Harmonized Tripartite Guideline Q2 (R1), 1994). Standard

solutions of decreasing concentration were produced by successive dilution of the

http://en.wikipedia.org/wiki/Measurement

http://en.wikipedia.org/wiki/Quantity

http://en.wikipedia.org/wiki/Value_%28mathematics%29

http://en.wikipedia.org/wiki/Reproducibility

http://en.wikipedia.org/wiki/Repeatability

http://en.wikipedia.org/wiki/Result

60

lowest calibration standard and the resulting solutions were injected in triplicate. A

volume or concentration of 0.01 ml, the least calibration standard concentration was

diluted 10 times.

3.1.8 Range and system suitability

The range was determined by observing the interval between the upper and lower

concentration of phenylephrine hydrochloride for which the HPLC analytical assay

has suitable level of linearity, accuracy and precision (ICH Harmonized Tripartite

Guideline Q2 (R1), 1994).

System suitability is a measure of the performance and chromatographic quality of

the total analytical system, namely instrumentation and procedure (ICH Harmonized

Tripartite Guideline Q2B, 1994). Six replicate injections of the standard solution of

phenylephrine hydrochloride were performed. The requirements for system suitability

are that the percentage relative standard deviation of the peak responses due to for

six injections must be less than or equal to 1.0 %. The tailing factor of the peak must

not be more than 2.0. A peak is labeled as tailing or asymmetrical when it deviates

from the ideal, symmetrical shape of a Gaussian peak. The later-eluted half of the

peak is wider than the front half and the broadening appears to be emphasized near

the baseline. Peak tailing is measured using the USP tailing factor (Tf) (Dolan,

2003). The tailing factor is expressed by: Tf = a + b

2a

where Tf = USP tailing factor

a = front half-width measured at 5 % of peak height

b = back half-width measured at 5 % of peak height (USP, 2004).

3.1.9 Specificity

Stress testing of API and finished product is undertaken to demonstrate specificity

when developing stability indicating methods (Reynolds et al., 2002). The API and

finished products were stressed in order to force phenylephrine hydrochloride

degradation. This would verify or exclude the presence of co-eluting impurities or

degradation products arising from the mobile phase, solvent, unstressed and

61

stressed products. The products are referred to as product I, II, III, IV, V and were

formulated as indicated in Table 5 (section 3.3.2). The nature of the specificity

samples and the relevant stress conditions are listed below:

All five products and the phenylephrine hydrochloride were subjected to the following

stress conditions after which they were analysed:

0.2 M NaOH for 30 minutes (reflux system).

0.2 M HCl for 30 minutes (reflux system).

0.2 M H2O2 for 30 minutes (reflux system).

UV lights (17 hours inside a stability chamber).

100 °C (24 hours inside a stability chamber).

65 °C (1 month inside a stability chamber).

40 °C / 75% RH (1 month inside a stability chamber).

Unstressed batch of phenylephrine hydrochloride and products I–V.

A quantity of 10 mg of unstressed phenylephrine hydrochloride was dissolved in 100

ml of dilution solvent and analysed using the HPLC method being validated. A

volume of 0.3 ml of unstressed products I–V was dissolved in 10 ml dilution solvent

and analysed using the HPLC method being validated.

A mass of 10 mg of phenylephrine hydrochloride was diluted to 100 ml with 0.2 M

HCl, 5 ml of this mixture was diluted to 25 ml with 0.2 M HCl and then refluxed for 30

minutes; the same manipulation was applied when using NaOH and H2O2. A

measured volume of 0.5 ml from product I–V was diluted to 100 ml with 0.2 M HCl

and refluxed for 30 minutes, 10 ml of this was diluted to 100 ml using the dilution

solvent, filtered and analysed using the HPLC method being validated.

Phenylephrine hydrochloride and products I–V were stressed in a stability chamber

(Binder, SA) which emitted both UV and visible light through a window glass filter

(type 2) and conformed to the requirements of the ICH guidelines. The stability

chamber had an irradiance level of 318 watts/m2 in order to expose the samples to

an overall illumination of not less than 1.2 million lux hours and an integrated near

62

UV energy of not less than 200 watt hours/m2 all according to ICH Harmonized

Tripartite Guideline Q1B, (2005).

All impurities and degradation products should be between 10 – 20 % of the API.

This includes samples stored under relevant stress conditions: light, heat, humidity,

acid/base hydrolysis and oxidation (ICH Harmonized Tripartite Guideline Q2 (R1),

2005). Degradants and impurities will be labeled as unknown (Ω) and if more than

one is present will be denoted as Ωi, Ωii, Ωiii and so on.

3.2 Determination of active–excipient compatibility

Active-excipients mixtures were achieved by mixing in a Schott beaker accurately

weighed 10 mg of phenylephrine hydrochloride and 10 mg of excipient, both were

dissolved in 10 ml ultra-pure water and transferred into plastic eye drop bottles. They

were stored at accelerated conditions for one month. A quantity of 5 ml was filtered

and analysed in triplicate using the validated HPLC method. The samples were

analysed before and after storage. The potency of the phenylephrine hydrochloride

was then calculated as a percentage of the initial potency. Significant change would

be defined as a 5% potency loss from the initial assay value of the API (Thakur et al.,

1999).

The assayed results of the active-excipient mixture were calculated on the

percentage of phenylephrine hydrochloride remaining in each sample from the linear

regression curve, y = 8541.1x + 438.55, taking the initial assay of phenylephrine

hydrochloride in each vial to be 100%. Physical stability was analyzed by visually

assessing the appearance and colour of the sample contents.

3.3 Manufacture of products

An important factor for eye drops is that they should be sterile when dispensed in a

multiple-application container. The preservative should be effective against

accidental introduction of micro-organisms and contaminants. The formulations were

manufactured in a clean environment in a laminar-flow hood using aseptic technique.

63

3.3.1 Materials

The hotplate magnetic stirrer utilized during the manufacturing process was a

Heidolph® MR 3002 (Labotec®, Midrand, SA), the storage chamber (BINDER® KMF

series, BINDER Inc, New York, USA) and the autoclave (Hirayama, New York,

USA), Natural dropper bottle 15 ml (LDPE) polyethylene container with closure

component (plugs with cap).Eeye drop bottles 13 MM CTRL dropper tip 0.031

needle natural (Amcor pharmaceutical packaging, New Jersey, USA), beakers and

graduated glass bottles (SCHOTT® North America, Inc. New York, USA), membrane

filter paper 0.45 µm (Millipore Corporation, Bedford, Massachusetts, USA), alcohol

preparation swabs WEBCOLTM, plastipak syringes (10 and 5 mL), HiCare int. latex

gloves large, avacare hypodermic needles (green) 21 g, alcohol 70% (particle free)

were all supplied by Alpha Pharm. Pty (Ltd), Port Elizabeth, South Africa.

3.3.2 Product manufacture

1 M Sodium Hydroxide: A mass of 40 g of sodium hydroxide was mixed in a round

bottom flask with one liter of purified water.

Hydroxypropyl methylcellulose (0.3%): A mass of 3 g of HPMC grade E5 was

dispersed in a beaker with 150 ml of purified water at 90 °C, when thoroughly

hydrated; 850 ml of ice water was added and stirred until a clear homogenous liquid

was formed.

Sodium carboxy methylcellulose (0.2%): A mass of 2 g of SCMC was dispersed in

a beaker with 150 ml of purified water at 90 °C, when thoroughly hydrated; 850 ml of

ice water was added and stirred until a clear homogenous liquid was formed.

The summary of the formulation excipients, API and concentration can be seen

below in Table 5.

64

Table 5: Formulation summary of active pharmaceutical ingredient and excipients used in the manufacturing of products I–V

Lot Size: 1L

Material Product I

Product II

Product III

Product IV

Product V

Phenylephrine Hydrochloride 10% w/v 10% w/v 10% w/v 10% w/v 10% w/v

Sodium citrate Dihydrate

0.1% w/v 0.1% w/v - - -

Sodium Metabisulfite

- 1% w/v - - -

Boric Acid - - - 1.9% w/v -

Benzalkonium Chloride

0.1% v/v 0.1% v/v - - -

Hydroxypropyl methylcellulose QS to

0.3% w/v 0.3% w/v - - 0.3% w/v

Methyl hydroxybenzoate - - 0.18% w/v - 0.18% w/v

Propyl hydroxybenzoate - - 0.02% w/v - 0.02% w/v

Sodium carboxy Methylcellulose to

- - - 0.2 % w/v -

Water Purified (RO) 10% v/v 10% v/v 10% v/v 10% v/v 10% v/v

Ethylenediaminetetraacetic Acid

- - - 0.1% w/v -

Glycerol to - - 100% v/v -

1 M Phosphoric acida

a

a

1 M Sodium Hydroxidei

i

i i

i and a – For pH adjustment only, ( - ) means it was absent from the formulation

3.3.2.1 Sterilization for heat sensitive API and exipients

The API and excipients were sterilized as follows: The heat sensitive PE was

steamed in a water bath at 100 °C for 30 minutes. Autoclave is not

recommended as phenylephrine hydrochloride has tendency to decompose

on heating.

All other equipments and excipients were autoclaved as follows: 121 °C for 15

minutes at 1 atm (200 kPa or 15 psi).

After sterilization the bottles containing products were shaken every 15

minutes until cool, to make sure HPMC and SCMC stayed fully mixed and

hydrated.

3.3.3 Manufacturing methods for products I–V

The manufacturing processes followed the same general method, with slight

variations in the processes based on certain excipeints. The few exceptions are seen

with the preservatives methyl and propyl hydroxybenzoate. Ingredients were added

65

sequentially to the main portion of solution while mixing with the glass rod. The

formulation was mixed until visually homogenous before addition of the next

ingredient. The methyl and propyl hydroxybenzoate were dissolved in ultra-pure

water with the aid of gentle heat and mixing with a glass rod. Addition of the

dissolved preservatives to the bulk solution was done to ensure complete

quantitative transfer of all preservatives to the bulk formulation. The solution was

then transferred to a calibrated plastic bottle, the dropper inserted and the bottle

capped. The stability of the formulations was determined in the primary plastic

packaging. The laboratory scale manufacturing processes for the five products

(products I–V) can be seen in figures 5 to 9.

Figure 5: Laboratory scale 1000 ml manufacturing process of product I

3) Filtered and pasteurized at

100 °C for 30 minutes in

water bath

ii) Sodium citrate dihydrate 0.1% w/v Benzalkonium chloride 0.1% v/v HPMC 0.3% w/v to 100%

iii) Disperse and mix with a glass

rod until all dissolve

iv) Autoclave mixture at 121 °C for

15 minutes

5) Aseptically and

quantitatively mixed

6) A volume of 15 ml was

aseptically and quantitatively

transferred to the calibrated

plastic eye-drop bottle which

was capped.

4) Cooled to room

temperature of 25 °C

1) Ultra pure water 10% v/v

2) Disperse and mix with a glass

rod until all dissolve i) Phenylephrine hydrochloride 10% w/v

66

Figure 6: Laboratory scale 1000 ml manufacturing process of product II

ii) Sodium citrate dihydrate 0.1% w/v Sodium metabisulfite 0.1% w/v HPMC 0.3 % w/v to 100%




15 minutes



water bath

1) Ultra pure water 10%v/v

4) Cooled to room


5) Aseptically and






was capped

2) Disperse and mix with a

glass rod until all dissolve i) Phenylephrine hydrochloride 10% w/v

67

Figure 7: Laboratory scale 1000 ml manufacturing process of product III

ii)Methyl hydroxy benzoate 0.18% w/v Propyl hydroxy benzoate 0.02% w/v Glycerol to 100% v/v




15 minutes


4) Cooled to room


5) Aseptically and






was capped.


glass rod until all dissolve i)Phenylephrine hydrochloride 10%

w/v


100 °C for 30 minutes in water

bath

68

Figure 8: Laboratory scale 1000 ml manufacturing process of product IV

ii) Boric acid 1.9% w/v EDTA 0.1% w/v SCMC 0.2% w/v to 100%

iii) Disperse and mix with a glass rod

until all dissolve

iv) Autoclave mixture at 121 °C for 15

minutes


%

4) Cooled to room


5) Aseptically and



100 °C for 30 minutes in water

bath

2) Disperse and mix with a glass

rod until all dissolve i) Phenylephrine hydrochloride 10%

w/v





was capped.

69

Figure 9: Laboratory scale 1000 ml manufacturing process of product V

3.4 Stability Tests

Testing for stability is crucial in product development. It usually involves at least two

stages: firstly, accelerated tests on a prototype and secondly, storage under

probable conditions of the manufactured product. In SA, the submission of stability

data is compulsory before sale is permitted, result from accelerated stability studies

are usually acceptable for this purpose (Zahn, 2008).

Stability studies on the dosage forms were conducted at specific temperatures and

relative humidities representing storage conditions experienced in the climatic zones

ii) Methyl hydroxy benzoate 0.18% w/v Propyl hydroxy benzoate 0.02% w/v HPMC 0.3% w/v to 100%

iii) Disperse and mix with a glass rod

until all dissolve


15 minutes

1)Ultra pure water 10% v/v

4) Cooled to room


5) Aseptically and






was capped.



water bath


glass rod until all dissolve i)Phenylephrine hydrochloride 10% w/v

70

of South Africa (climatic zones I and II). The formulations were stored at 40

°C/75%RH, 25 °C/40%RH and 30 °C/65%RH. The five formulations were stored at

all three of the above mentioned conditions and tested in triplicate at 0, 3, and 6

months. The most appropriate and stable formulation was chosen based on the level

of degradation of the phenylephrine hydrochloride, pH change and appearance.

3.5 Qualitative and quantitative analysis of the formulations

3.5.1 Appearance and pH

The bulk appearance of the prepared products was visually examined for colour,

homogeneity, and for the appearance of any precipitate. The pH of the solutions

was measured using a pH/mV/ °C meter (744 pH meter, metrohm). Each solution

was stirred with a magnetic stirrer (Hanna instruments supplied by Tecnilab, SA) for

30 seconds before pH measurement.

3.5.2 Phenyleprine hydrochloride concentration

The unknown peaks have to be less than or equal to 1% while the phenylephrine

hydrochloride left must be between 95%–105% to be accepted as a successful

formulation. The mean peak area was divided by the theoretical amount of drug

found in the formulation multiplied as a percentage to give the final result of

phenylephrine hydrochloride left.

3.6 Test for preservative efficacy

The test challenged the formulation in its final primary container, with a prescribed

inoculum of suitable micro-organisms, storing the inoculated product at a prescribed

temperature, withdrawing the samples from the container at specified intervals of

time and counting the organisms in the samples removed. The two methods used

for determining bacterial numbers were the standard (viable) plate count method and

spectrophotometric (turbidimetric) analysis. The standard plate method reveals

information related to live bacteria while indirectly measuring cell density. The

71

spectrophotometric method is based on turbidity and indirectly measures all bacteria

(cell biomass), dead or alive (Harley and Prescott, 1998).

3.6.1 Procedure for standard plate count

Materials: Cultures of Escherichia coli (ATCC No. 38218), Candida albicans (ATCC

No. 66027), Pseudomonas aeruginosa (ATCC No. 27853), Staphylococcus aureus

(ATCC No. 43300) were obtained from the Department of Biomedical Technology at

the Nelson Mandela Metropolitan University. The bacteria were chosen because of

their classification as well as pathogenicity. Pipettes, petri plates, curvettes

(Hellma®, precision cells, Lasec, Johannesburg, SA) were obtained from Lasec

(Johannesburg, SA). Bunsen burner, platinum loop wire dispensers (1/200 mL)

(Lasec, Johannesburg, SA), water was produced by Ultra Clear TWF/El-Ion® system

which was pre-treated and made ultrapure (reverse osmosis) (Separations,

Johannesburg, SA). Tryptone soya broth and agar, Sabouraud broth and agar were

supplied by Sigma-Aldrich (Pty) Ltd (Kempton Park, SA). Sodium chloride was

obtained from Merck Laboratory Supplies (Pty) Ltd (Midrand, SA). Hockey stick was

obtained from Lasec (Johannesburg, SA). Tecam® SB-16 Shaking Water bath was

obtained from Spellbound Laboratory solutions (Port Elizabeth, SA). Vacutec

(Johannesburg, SA) supplied the P Spectra colony counter, Shimadzu® UV

spectrophotometer (UV-1800) (Shimadzu, Tokyo, Japan).

Media preparations: All broths and agars poured into 1000 ml Schott bottles with a

screw cap and autoclaved at 121 °C for 15 minutes. Tryptone soya broth was made

by weighing 25 g and dissolving 1000 ml of distilled water. Tryptone soya agar was

made by weighing 40 g which was dissolved in 1000ml of distilled water. Sabouraud

broth was made by weighing 30 g which was dissolved in 1000 ml of distilled water.

Sabouraud agar was made by weighing 45 g which was dissolved in 1000 ml of

distilled water.

3.6.2 Procedure for plating the bacteria and fungi

All Petri dishes and saline bottles were labelled accordingly. Using aseptic

technique, 0.1 ml was added to a 9.9 ml sterile saline and was shaken vigorously to

72

distribute the bacteria. A volume of 0.1 ml of this was aseptically transferred to a

second 9.9 ml sterile saline, it was then capped and the second dilution was shaken

vigorously. The process was repeated until 10-8 dilution was attained. Volumes of 0.1

ml and 0.01 ml from each dilution of microbes were aseptically transferred to petri

plates. The tryptone soya agar at 48 °C was aseptically poured into a petri dish. The

agar and sample were immediately mixed by gently moving the plate in a figure-eight

motion. The process was repeated for the other plates which were then left to cool;

they were inverted and incubated at 35 °C for 24 hours. The plates were counted

using the P Spectra® colony counter. Plates with more than 250 colonies were not

counted and were labelled too numerous to count (TNTC). Plates with fewer than 25

colonies were designated too few to count (TFTC). The colony forming unit per

millilitre was calculated by dividing the number of colonies by the dilution factor

(Harley and Prescott, 1998).

3.6.3 Standardization of cultures using turbidimetry method

One empty tube and five tubes containing 3 ml of sterile tryptone soya broth were

placed in a test-tube rack and labelled A–E, with the exception of the empty tube.

The five tubes of broth were used to make five serial dilutions of the culture. With the

aid of a spectrophotometer (Shimadzu® UV spectrophotometer (UV-1800) and

curvettes (Hellma®, precision cells) the different cultures were standardized at a

wavelength of 600 nm; the blank was a sterile broth without the culture. From the

culture broth, different absorbance concentrations of 0.200 to 1.0 were achieved with

sterile saline using the serial dilution method. Each absorbance concentration was

plated and the colony counted in triplicate. A straight line graph was constructed by

plotting the absorbance of the culture broth at 600 nm versus the number of colony

forming units, and a linear regression trendline was fitted to the plot (Microsoft

Excel® 2007, Microsoft Corporation). A linear relationship was found with all the

organisms, as indicated by the linear correlation coefficient (R2), which was greater

than 0.99, even though the y-intercepts were significantly different to zero (Harley

and Prescott, 1998). This method was applied to the different microorganism.

73

3.6.4 Preservative efficacy

The preservative properties of the formulation are adequate if, in the conditions of

the test, there is a significant fall or no increase in the number of micro-organisms in

the inoculated product after the times and at the temperatures prescribed. Negative

controlled samples were prepared by excluding preservatives, and inoculums of 100

µl of 106 CFU/ml were added to both control and products I–V. The inoculated

samples were stored away from light at 25 °C for 6 hours, 24 hours, 7 days, 14 days

and 28 days. Aliquots of 1 ml were removed from each sample at zero, six and 24

hours and after 7, 14 and 28 days intervals. The sampled were then enumerated.

One milliliter aliquots were transferred to a sterile 10 ml nutrient broth and plated in

duplicate on tryptone soya agar (for bacteria) or Sabouraud dextrose agar (for fungi).

Plates were incubated at 35 °C for 48 hours for bacteria and 25 °C for 72 hours for

fungi. Raw data counts were converted to log10 values and the reduction from

inoculum values was calculated for evaluation against compendial requirements

found in Table 6 below.

Table 6: Criteria for tested microorganisms (USP, 2004)

Category 1 products such as eye drops

Bacteria Not less than 1.0 log reduction from the initial calculated count at 7 days, not less than 3.0 log reduction from the initial count at 14 days, and no increase from the 14 days' count at 28 days.

Yeast and molds

No increase from the initial calculated count at 7, 14, and 28 days.

The samples were diluted in a 1:10 ratio at the time of testing, 10 CFU (or 1.0 log

reduction) is the lowest sensitivity allowed by the test.

3.7 Determination of viscosity

3.7.1 Equipments and method

All measurements were performed with an Anton Paar RheolabQC rheometer with

cylinder measuring CC27 according to ISO 3219. ISO 3219 describes the

dimensions of the cylinder geometry and defines the ratio of measuring cup radius to

measuring bob radius as 1.0847. This guarantees an industrial standard for shearing

74

the sample in the measuring gap, independent of the measuring system size and

manufacturer. The temperature of the measuring system was controlled using a

thermostat (Anton Paar ViscoTherm VT2) directly connected to the Antor Paar

rheometer (Šklubalova & Zatloukal, 2011). For all measurements temperature was

23 °C this was kept constant as determination of the viscosity η and yield point is

very dependent on the temperature.

The testing conditions for the determination of the yield point were as follows:

1. Preshearing with = 5 s-1 over t = 1 minute to homogenise and control the

temperature of the product.

2. Rest phase with = 0 s-1 over t = 30 seconds. Intervals 1 and 2 increase the

reproducibility of the measurements.

3. Shear stress ramp of = 1 Pa to 30 Pa with 200 measuring points per 1s

(duration t = 200 seconds).

3.8 Statistical analysis

Descriptive and inferential statistics were used. Standard deviation (+/-) and mean

were calculated from measured replicates using the software package GraphPad

Prism version 6.01 (GraphPad Software Inc., San Diego, USA). Comparisons of

means were achieved using one-way ANOVA. Statistically significant differences

were tested using Student’s t-test with significance in difference defined as p<0.05.

The experimental design was based on a non-matching or pairing of mean, with the

mean of each observation being compared with the mean of every other observation.

The Bonferroni test was chosen as it corrected for multiple comparisons and

indicated confidence intervals and significance.

75

4. RESULTS AND DISCUSSION

4.1 Validation of the stability indicating assay

The method used to analyse the formulations was obtained from the USP. Though it

was a pharmacopoeial method its suitability for use on the present system had to be

validated. The validation process followed ICH guidelines and the results are

indicated below.

4.1.1 Linearity

Linearity is determined by a calibration curve which was constructed by plotting the

area of the phenylephrine hydrochloride peak versus phenylephrine hydrochloride

concentration. Figure 10 below shows linearity over the concentration range of

0.0125 to 0.15 mg/ml. The linear regression equation was y = 8541.1x + 438.55, with

a correlation coefficient, R2, equal to 0.9999. The requirements for linearity were

attained, as the correlation coefficient of the regression line was greater than 0.999

and the percentage relative standard deviations for the phenylephrine hydrochloride

peak areas of multiple injections 0.0125, 0.025, 0.05, 0.075, 0.1 and 0.15 mg/ml

were all less than 1.5%. This means that the results achieved are directly

proportional to the concentration of phenylephrine hydrochloride within a given

range. 8541.1 defines the slope, y, is 438.55.

Figure 10: Graph showing a mean peak area versus concentration of replicate samples of phenylephrine hydrochloride standards. Linear regression equation: y = 8541.1x + 438.55, R

2 =

0.9999.

.

0

200000

400000

600000

800000

1000000

1200000

1400000

0 0.05 0.1 0.15 0.2

pe

ak

are

a

concentration (mg/ml)

linearity graph of phenylephrine hydrochloride standards

76

4.1.2 Accuracy

Accuracy of the method was within acceptable range as indicated in Table 7 below.

The percentage relative standard deviations calculated for the samples at the lower,

middle and upper limits of the concentration range were all below 0.5%. This means

by applying the analytical method of a known purity concentration (linearity) and

comparing the results of accuracy the differences were not greater than 0.5%.

Table 7: Accuracy data for quantification of phenylephrine hydrochloride

Theoretical

Concentration

(mg/ml)

Actual Concentration,

Mean (n = 3)

(mg/ml)

Relative Standard

Deviation

(% RSD)

Percentage

Recovery (%)

9.5 9.46 0.36 99.5

54 54.1 0.02 100.1

138 136.89 0.05 99.1

The recovery percentage for the samples was between the limits of 99.00 to

100.10%.

4.1.3 Precision

Precision was within acceptable range as seen below in Table 8. The percentage

relative standard deviations calculated for the samples at the lower, middle and

upper limits of the concentration range were all below 0.5%. The recovery

percentage for the samples was between the limits of 99.00 to 100.00%. The method

being validated showed precision was achieved as the degree of agreement among

individual test results was demonstrated by the relative standard deviation.

Table 8: Precision data for quantification of phenylephrine hydrochloride

Theoretical Concentration (mg/ml)

Actual Concentration, Mean (n=3) (mg/ml)

Relative Standard Deviation (% RSD)

Percentage Recovery (%)

9.5 9.48 0.42 99.8

54 53.86 0.06 99.7

138 137.29 0.16 99.4

77

4.1.4 Limit of detection (LOD) and quantification (LOQ)

The LOD was determined by serial dilutions of the lowest concentration (0.0125

mg/ml) of the attained phenylephrine hydrochloride standard and found to be 12.3

μg/ml. The amount stated is the lowest amount of phenylephrine hydrochloride in a

sample that can be detected. The LOD is the lowest concentration for which the

relative standard deviation of multiple injections is less than 5.0%. The LOQ was

found to be 41 μg/ml. The amount shows the lowest amount of analyte in a sample

that can be determined with acceptable precision and accuracy. By convention, the

LOD value is taken as 0.3 times the LOQ (Armbruster & Pry, 2008) while LOQ = 3.33

LOD (Thomsen et al., 2003). The results of the two did fit this mathematical

assumption. These methods are HPLC specific and are dependent on the type of

HPLC conditions; therefore re-determination in each laboratory is necessary. The

range was 0.0125 to 0.15 mg/ml.

4.1.5 Specificity and system suitability

Specificity of a chromatographic method is the ability of the method to accurately

measure the analyte response in the presence of all potential sample components.

Specificity is useful to show that an analyte response cannot be attributed to more

than one component (Rozet et al., 2011). The chromatograms were examined for the

presence of compounds, metabolites, impurities, degradants and matrix components

that may interfere or partly co-elute with the phenylephrine hydrochloride peak.

The mean peak area for the 6 replicate injections of phenylephrine hydrochloride had

a percentage relative standard deviation of 0.14 %. The tailing factor was 2.0. The

analytical system thus complied with the requirements specified by the system

suitability as the percentage relative standard deviation of the peak responses due to

phenylephrine hydrochloride for six injections was less than 1.0 %. The column was

thus deemed suitable for analysis. There is a peak tailing as potrayed in the

chromatograms, some reasons could be due to more than one retention mechanism

is present in a separation, and one of those mechanisms is overloaded. At the

surface, the polymer terminates in silanol groups. There are different possible silanol

configurations which are abundant at the surface of the columns as free silanols, but

78

silicon atoms with two hydroxyl groups in a geminal configuration also are present. If

the silanol groups are positioned next to each other, an associated silanol group can

share a proton with an adjacent group. The free silanol groups are more acidic than

the geminal and associated groups, and they interact more strongly with basic

solutes. The result is peak tailing commonly associated with the separation of bases.

To further complicate matters, trace metals (e.g., iron and aluminium) can be present

in the silica matrix. These trace metals can act as ion-exchange sites or, when

adjacent to free silanols, can withdraw electrons, which makes the silanols even

more acidic. To reduce the incidence of tailing or fronting, concentrations should be

reduced proportionally by appropriate dilutions (Dolan, 2003).

Mobile phase chromatogram showed no interference as it produced a chromatogram

which had a steady baseline and no ghost peaks as seen in Figure 11.

Figure 11: HPLC Chromatogram for mobile phase alone.

The peak produced by phenylephrine hydrochloride shows no interference from

contaminants or impurities. Figure 12 below shows it eluted after 7.8 minutes and

had an average retention time of 7 minutes. This means the conditions and

concentrations were suitable for the phenylephrine hydrochloride standard. The

purity peak shows the phenylephrine hydrochloride standard was pure as the value

was 1.0, the closer the value is to one the purer a compound is. The start of the

mobile phase for the gradient elution was different from the dilution solvent; as a

result a constant peak is noted between 2.5 and 3.5 minutes as seen in the figures

below. They are known as ghost or false peaks. It takes that amount of time for the

injected solvent to reach the detector and the polarity differences caused by the

changes in pH and solvent phase causes it to be identified as a peak. The peaks

were not symmetrical and fronting or tailing was found to be 2.0.

79

Figure 12: HPLC Chromatogram for phenylephrine hydrochloride dissolved in mobile phase with a retention time of 7.80 minutes.

Figure 13: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999.

Products I–V without any stress are indicated below in figures 14–23. They show

purity indexes of 1.0 and chromatograms showing no co-elution or impurities. The

average retention time for PE was 7.85 minutes.

Figure 14: HPLC Chromatogram for product I dissolved in mobile phase with a retention time of 7.87 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak

Zero LinePurity Curve

0.0 2.5 5.0 7.5 10.0 12.5 min

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

mAU280nm,4nm (1.00)

80

Figure 15: Peak purity profile calculated using PDA data (from 190–800 nm) for Product I prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999054

Figure 16: HPLC Chromatogram for product II dissolved in mobile phase with a retention time of 7.82 minutes.

Figure 17: Peak purity profile calculated using PDA data (from 190–800 nm) for Product II prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.996482.

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak


81

Figure 18: HPLC Chromatogram for product III dissolved in mobile phase with a retention time of 7.83 minutes.

Figure 19: Peak purity profile calculated using PDA data (from 190–800 nm) for Product III prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.998178.

Figure 20: HPLC Chromatogram for product IV dissolved in mobile phase with a retention time of 7.89 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50

mAU280nm,4nm (1.00)

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

82

Figure 21: Peak purity profile calculated using PDA data (from 190–800 nm) for Product IV prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.996947.

Figure 22: HPLC Chromatogram for product V dissolved in mobile phase with a retention time of 7.84 minutes.

Figure 23: Peak purity profile calculated using PDA data (from 190–800 nm) for product V prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999587.

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak


83

Stress under UV light. Products I–V were stressed using UV light, their

chromatograms did not show interference or co-elution with phenylephrine

hydrochloride. Figures 24–35 below indicate that the excipients did not react with the

phenylephrine hydrochloride and no degradants co-eluted with the phenylephrine

hydrochloride. The plastic bottle did not change in colour as the solutions were still

clear and homogenous. The average retention time for the products was 7.85

minutes. Peak purity index was 1.0 for products I, II, IV and V. Product III had a peak

purity index of 0.999999.

Figure 24: HPLC Chromatogram for phenylephrine hydrochloride stressed under UV light dissolved

in mobile phase with a retention time of 7.81 minutes.

Figure 25: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak


84

Figure 26: HPLC Chromatogram for product I stressed under UV light dissolved in mobile phase with a retention time of 7.86 minutes.

Figure 27: Peak purity profile calculated using PDA data (from 190–800 nm) for product I prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999054.

Figure 28: HPLC Chromatogram for product II stressed under UV light dissolved in mobile phase with a retention time of 7.82 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0

10

20

30

40

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60

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

85

Figure 29: Peak purity profile calculated using PDA data (from 190–800 nm) for product II in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999116.

Figure 30: HPLC Chromatogram for product III stressed under UV light dissolved in mobile phase with a retention time of 7.85 minutes.

Figure 31: Peak purity profile calculated using PDA data (from 190–800 nm) for product III in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.997116.

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

5.0

10.0

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20.0

25.0

30.0

mAUPeak


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15

20

25

30

35

40

45

50

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


86

Figure 32: HPLC Chromatogram for product IV stressed under UV light dissolved in mobile phase with a retention time of 7.81 minutes.

Figure 33: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV in mobile

phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point

threshold = 0.997916.

Figure 34: HPLC chromatogram for product V stressed under UV light dissolved in mobile phase with a retention time of 7.87 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

87

Figure 35: Peak purity profile calculated using PDA data (from 190–800 nm) for product V in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.997475.

No colour change was observed with products I–V and phenylephrine hydrochloride

remained stable under the UV light stress. If it was unstable a colour chang would be

observed from clear to dark brown. The average concentrations of the products

remained at 99.01%. Using the above mentioned statistical analysis (one-way

ANOVA), there were no significant difference in the API concentration among the

products (I–V) manufactured at time zero (T0). They had a P-value of p > 0.9999, this

meant that the products during formulation and testing maintained their API at T0.

Physical and chemical degradation are known to occur in phenylephrine

hydrochloride and this is sometimes accompanied by a change in color, e.g.

changing from a white or almost white colour into a darker, brownish color.

Discoloration is accelerated by light, but it occurs eventually even in light-protected

solutions. Degradation of phenylephrine hydrochloride may be caused by a variety of

factors including the presence of oxygen, moisture, reducing sugars, bases and high

temperature (Douša et al., 2011)

Stress with HCl: Products I–V were stressed with HCl, their chromatograms did not

show interference or co-elution with phenylephrine hydrochloride. Figures 36–47

below indicates that the excipients did not react with the phenylephrine hydrochloride

and no degradants co-eluted with the phenylephrine hydrochloride. Discolorations

were observed during the reflux of products I–V with HCl, the colours ranged from

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


88

very light yellow to yellow. The greatest change was observed in product III, as it

turned bright yellow. These could mean glycerol, HCl and phenylephrine

hydrochloride were undergoing a reaction of oxidation or reduction. The average

retention time for the products was 7.85 minutes. Peak purity index was 1.0 for

products I–V however; the average API concentration had reduced to 79.64 %.

Figure 36: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.86 minutes.

Figure 37: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999574.

Figure 38: HPLC chromatogram of Product I stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.82 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

89

Figure 39: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.997116.

Figure 40: HPLC chromatogram for product II stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.83 minutes.

Figure 41: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.996296.

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

5.0

10.0

15.0

20.0

25.0

30.0

mAUPeak


90

Figure 42: HPLC chromatogram for product III stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.91 minutes.

Figure 43: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed

with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index

=0.999999; Single point threshold = 0.995179.

Figure 44: HPLC chromatogram for product IV stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.86 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

5.0

10.0

15.0

20.0

25.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

91

Figure 45: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed

with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index

=1.000000; Single point threshold = 0.997097.

Figure 46: HPLC chromatogram of product V stressed with 0.2 M HCl dissolved in mobile phase with

a retention time of 7.80 minutes.

Figure 47: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =1.000000; Single point threshold = 0.999578.

Stress with NaOH: Products I–V were stressed with NaOH, their chromatograms

did not show interference or co-elution with phenylephrine hydrochloride. Figures

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0

10

20

30

40

50

60

mAUPeak


92

48–59 below indicate that the excipients did not react with the phenylephrine

hydrochloride nor did degradation products of products I–V co-elute with the

phenylephrine hydrochloride. Discolorations were observed during the reflux of

products I–V with NaOH, the colours changed from clear to brown. The average

retention time for the products was 7.84 minutes. Due to the caustic nature of NaOH

the products had a reduced average API concentration of 42.36%.

Figure 48: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M NaOH dissolved

in mobile phase with a retention time of 7.82 minutes

Figure 49: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =0.999999; Single point threshold = 0.996847.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

5.0

10.0

15.0

20.0

25.0

30.0

mAUPeak


93

Figure 50: HPLC chromatogram of product I stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.89 minutes.

Figure 51: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =1.000000; Single point threshold = 0.995815.

Figure 52: HPLC chromatogram of product II stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.9 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

5.0

10.0

15.0

20.0

25.0

30.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

94

Figure 53: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996370.

Figure 54: HPLC chromatogram for product III stressed with 0.2 M NaOH dissolved in mobile phase

with a retention time of 7.79 minutes.

Figure 55: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.998771.

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

37.5mAU

280nm,4nm (1.00)

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

2.5

5.0

7.5

10.0

12.5

15.0

mAUPeak


95

Figure 56: HPLC chromatogram of product IV stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.91 minutes.

Figure 57: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed

with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =

0.999999; Single point threshold = 0.996930.

Figure 58: HPLC chromatogram for product V stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.85 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45mAU

280nm,4nm (1.00)

96

Figure 59: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999587.

Stress with H2O2: Products I–V were stressed with H2O2, their chromatograms did

not show interference or co-elution with phenylephrine hydrochloride. Figures 60–71

below indicates that the excipients did not react with the phenylephrine hydrochloride

or produce degradants found in products I–V which co-eluted with the phenylephrine

hydrochloride. Discolorations were observed during the reflux of products I–V with

H2O2, the colours changed from clear to pale yellow (products I, II, IV and V) and

from clear to lime green (product III). The average retention time for the products

was 7.82 minutes. Due to the oxidizing nature of H2O2 the products had a reduced

average API concentration of 26.43%.

Figure 60: HPLC chromatogram for phenylephrine hydrochloride stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.82 minutes.

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

5.0

10.0

15.0

20.0

25.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50

55

mAU280nm,4nm (1.00)

97

Figure 61: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999987.

Figure 62: HPLC chromatogram for product I stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.81 minutes.

Figure 63: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999522.

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

2.5

5.0

7.5

10.0

12.5

15.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50

55

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

2.5

5.0

7.5

10.0

12.5

15.0

mAUPeak


98

Figure 64: HPLC chromatogram for product II stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.9 minutes.

Figure 65: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed

with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =

0.999999; Single point threshold = 0.999722.

Figure 66: HPLC chromatogram of product III stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time 7.94 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50

55

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

-5

0

5

10

15

20

25

30

35

40

45

50

55

60mAU

280nm,4nm (1.00)

99

Figure 67: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999706.

Figure 68: HPLC chromatogram for product IV with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.81 minutes.

Figure 69: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999896.

7.75 8.00 8.25 min

0.00

0.25

0.50

0.75

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50

55

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.0

2.5

5.0

7.5

10.0

12.5

mAUPeak


100

Figure 70: HPLC chromatogram for product V stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.88 minutes.

Figure 71: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999536.

Stress with heat and humidity: Products I–V were stressed with heat and humidity;

their chromatograms did not show interference or co-elution of impurities with

phenylephrine hydrochloride. Figures 72–98 below indicates that the excipients did

not react with the phenylephrine hydrochloride. In Figure 72, phenylephrine

hydrochloride stressed at 100 °C for 24 hours in a stability chamber showed a

reduction of its API to 95% while showing no physical discolorations. Heat therefore

has an effect in reducing the potency of phenylephrine hydrochloride.

Chromatograms of products I–V stressed at the 65 °C for one month are shown in

figures 74–85 below. The API concentration was reduced and products I and III

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50mAU

280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


101

showed degardants or impurities eluting at 2, 5 and 10 minutes. Tailing was

observed in the all products stressed under this condition, which could mean the API

had reduced however the peak purity profile indicated that it was still pure.

Degradants did not interfere or co-elute with phenylephrine hydrochloride. Changes

in physical appearance were recorded in Table 9 below.

Table 9: Physical appearance of phenylephrine hydrochloride and products I–V before and after storage conditions 40 °C/75%RH for 1 month

Item Initial Physical appearance

Physical appearance After 4 weeks of storage

40 °C/75%RH 65 °C

Phenylephrine Hydrochloride

Clear solution, no precipitate/residue, no colour change

No change in initial appearance

Yellow, no precipitate formed

Product I Clear solution, no precipitate/residue, no colour change


Light yellow, no precipitate formed

Product II Clear solution, no precipitate/residue, no colour change


Tinge brown, No precipitate formed

Product III Clear solution, no precipitate/residue, no colour change


Slight yellow, No precipitate formed

Product IV Clear solution, no precipitate/residue, no colour change



Product V Clear solution, no precipitate/residue, no colour change



Bold print indicates physical mixtures that showed a change in appearance after 4 weeks of storage

Table 9 above shows changes in appearance at 65 °C for all products. The same did

not occur at the 45 °C/75%RH storage condition where none of the samples

underwent any physical change.

Chromatograms of products I–V stressed at the 40 °C/75%RH for one month are

indicated in figures 86–97 below. The API concentration was reduced, however all

concentrations were above the 95%, except for product V which was at 94.97%.

Products I–V were able to withstand the heat and humidity without showing any

change in physical appearance. Tailing was observed in the all products stressed

under this condition, which could mean the API had slightly reduced however the

peak purity profile indicated that it was still pure. Degradants did not interfere or co-

elute with phenylephrine hydrochloride.

102

Figure 72: HPLC chromatogram for phenylephrine hydrochloride stored at 100 °C for 24 hours dissolved in mobile phase with a retention time of 7.8 minutes.

Figure 73: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stored at 100 °C for 24 hours in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.5 8.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

10

20

30

40

50

60

mAUPeak


103

Figure 74: HPLC chromatogram for phenylephrine hydrochloride stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.83 minutes.

Figure 75: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999989.

Figure 76: HPLC chromatogram for product I stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.92 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.0

5.0

10.0

15.0

20.0

25.0

30.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

104

Figure 77: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stored at

65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.999956.

Figure 78: HPLC chromatogram for product II stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.84 minutes.

Figure 79: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999056.

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50

55

60

mAU280nm,4nm (1.00)

7.75 8.00 8.25 min

0.0

0.1

0.2

0.3

0.4

0.5

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


105

Figure 80: HPLC chromatogram for Product III stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.89 minutes.

Figure 81: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999720.

Figure 82: HPLC chromatogram for Product IV stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.87 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50

55

mAU280nm,4nm (1.00)

7.75 8.00 min

0.0

0.1

0.2

0.3

0.4

0.5

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

37.5

40.0

mAU280nm,4nm (1.00)

106

Figure 83: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =0.999999; Single point threshold = 0.999803.

Figure 84: HPLC chromatogram for Product V stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.90 minutes.

Figure 85: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999752.

7.75 8.00 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

5.0

10.0

15.0

20.0

25.0

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Figure 86: HPLC chromatogram for phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.86 minutes.

Figure 87: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999056.

Figure 88: HPLC chromatogram for Product I stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.87 minutes.

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108

Figure 89: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stored at 40 °C/75% RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.995566.

Figure 90: HPLC chromatogram for Product II stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.84 minutes.

Figure 91: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999579.

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Figure 92: HPLC chromatogram for product III stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.81 minutes.

Figure 93: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.994609.

Figure 94: HPLC chromatogram for Product IV stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.85 minutes.

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280nm,4nm (1.00)

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110

Figure 95: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.994609.

Figure 96: HPLC chromatogram for Product V stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.83 minutes.

Figure 97: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.995332.

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Peak purities of all samples of stressed and unstressed phenylephrine hydrochloride

and finished product solutions are indicated in Table 10 below. Phenylephrine

hydrochloride alone showed loss to the combination of heat and humidity.

Table 10: Results showing absence of impurity from a series of stressed and unstressed samples of phenylephrine hydrochloride (API) and products.

Number Sample name Co-eluting impurities found

Image reference

1 Mobile phase only None Figure 11

2 Active: unstressed None Figure 12

3 Product I, unstressed None Figure 14

4 Product II, unstressed None Figure 16

5 Product III, unstressed None Figure 18

6 Product IV, unstressed None Figure 20

7 Product V, unstressed None Figure 22

8 Active, stressed under UV for 17 hours

None Figure 24

9 Product I, stressed under UV for 17 hours

None Figure 26

10 Product II, stressed under UV for 17 hours

None Figure 28

11 Product III, stressed under UV for 17 hours

None Figure 30

12 Product IV, stressed under UV for 17 hours

None Figure 32

13 Product V, stressed under UV for 17 hours

None Figure 34

14 Active, stressed with HCl None Figure 36

15 Product I, stressed with HCl None Figure 38

16 Product II, stressed with HCl None Figure 40

17 Product III, stressed with HCl None Figure 42

18 Product IV, stressed with HCl None Figure 44

19 Product V, stressed with HCl None Figure 46

20 Active, stressed with NaOH None Figure 48

21 Product I, stressed with NaOH None Figure 50

22 Product II, stressed with NaOH None Figure 52

23 Product III, stressed with NaOH None Figure 54

24 Product IV, stressed with NaOH None Figure 56

25 Product V, stressed with NaOH None Figure 58

26 Active, stressed with H2O2 None Figure 60

27 Product I, stressed with H2O2 None Figure 62

28 Product II, stressed with H2O2 None Figure 64

29 Product III, stressed with H2O2 None Figure 66

30 Product IV, stressed with H2O2 None Figure 68

31 Product V, stressed with H2O2 None Figure 70

32 Active, stressed at 100 °C None Figure 72

33 Active, stressed at 65 °C None Figure 74

34 Product I, stressed at 65 °C None Figure 76

35 Product II, stressed at 65 °C None Figure 78

36 Product III, stressed at 65 °C None Figure 80

37 Product IV, stressed at 65 °C None Figure 82

38 Product V, stressed at 65 °C None Figure 84

39 Active, stressed at 40 °C/75%RH None Figure 86

112

40 Product I, stressed at 40 stressed at 40 °C/75%RH

None Figure 88

41 Product II, stressed at 40 °C/75%RH

None Figure 90

42 Product III, stressed at 40 °C/75%RH

None Figure 92

43 Product IV, stressed at 40 °C/75%RH

None Figure 94

44 Product V, stressed at 40 °C/75%RH

None Figure 96

The products were decomposed (reduction in API concentration) by the combination

of heat and humidity, fairly decomposed by heat, and significantly broken (p<0.05)

down by hydrogen peroxide and base as seen in Table 11.

Table 11: Results showing phenylephrine hydrochloride left with samples stressed and unstressed (API and Products)

Sample number Sample condition Phenylephrine hydrochloride left

2 Active, unstressed 99.57%

3 Product I, unstressed 100.20%

4 Product II, unstressed 97.09%

5 Product III, unstressed 97.20%

6 Product IV, unstressed 98.68%

7 Product V, unstressed 97.31%

8 Active, stressed under UV light 99.10%

9 Product I, stressed under UV light 99.10%

10 Product II, stressed under UV light 99.61%

11 Product III, stressed under UV light 99.66%

12 Product IV, stressed under UV light 98.33%

13 Product V, stressed under UV light 98.63%

14 Active, stressed with HCl 75.26%

15 Product I, stressed with HCl 77.67%

16 Product II, stressed with HCl 81.11%

17 Product III, stressed with HCl 78.43%

18 Product IV, stressed with HCl 82.18%

19 Product V, stressed with HCl 83.21%

20 Active, stressed with NaOH 33.86%

21 Product I, stressed with NaOH 43.11%

22 Product II, stressed with NaOH 42.56%

23 Product III, stressed with NaOH 30.29%

24 Product IV, stressed with NaOH 50.77%

25 Product V, stressed with NaOH 53.57%

26 Active, stressed with H2O2 23.73%

27 Product I, stressed with H2O2 26.79%

28 Product II, stressed with H2O2 26.29%

29 Product III, stressed with H2O2 32.42%

30 Product IV, stressed with H2O2 23.55%

31 Product V, stressed with H2O2 25.80%

32 Active, stressed at 100 °C 95.42%

113

33 Active, stressed at 65 °C 92.42%

34 Product I, stressed at 65 °C 86.92%

35 Product II, stressed at 65 °C 90.74%

36 Product III, stressed at 65 °C 91.47%

37 Product IV, stressed at 65 °C 80.52%

38 Product V, stressed at 65 °C 88.21%

39 Active, stressed at 40 °C/75%RH 90.20%

40 Product I, stressed at 40 °C/75%RH 96.57%

41 Product II, stressed at 40 °C/75%RH 98.48%

42 Product III, stressed at 40 °C/75%RH 95.99%

43 Product IV, stressed at 40 °C/75%RH 95.37%

44 Product V, stressed at 40 °C/75%RH 94.97%

The HPLC method used for the phenylephrine hydrochloride and finished products

was found to be suitable because the degradants showed no interference. The range

retention time for phenylephrine was 7.78–7.92 minutes. The impurities did not

interfere or co-elute with phenylephrine hydrochloride, as its peak was distinct and

clear.

4.2 Active and excipient study

Three replicates of active and excipient mixtures at a ratio of 1:1 were stored at 40

ºC/75%RH in plastic containers for three months. They were then assayed using the

HPLC method. The percentage of phenylephrine hydrochloride remaining in each

sample was calculated from the linear regression curve, y = 8541.1x + 438.1 taking

the initial quantity of phenylephrine hydrochloride in each container to be 100%. The

potency of samples stored at 40 ºC/75%RH and the unstressed samples were within

95–105%. Figures 98–121 show the chromatograms of phenylephrine hydrochloride

alone and phenylephrine hydrochloride:combined with excipients; there was no

interference observed between the phenylephrine hydrochloride and excipients.

Table 12 below shows that all the samples were within the required potency range of

95–105%.

Table 12: Assay result showing phenylephrine hydrochloride and excipients in a 1:1 ratio after storage conditions 40 °C/75%RH for 1 month

Phenylephrine hydrochloride and excipients Percentage of phenylephrine hydrochloride

(API (left) % ± % RSD)

40 °C / 75 % RH

Phenylephrine hydrochloride alone 98.15 ± 0.41

Boric acid 99.48 ± 1.53

Benzalkonium Chloride 98.63 ± 0.25

EDTA 97.73 ± 0.48

114

Glycerol 95.79 ± 0.22

Methyl Paraben 96.33 ± 1.86

Propyl Paraben 96.73 ± 0.36

Sodium metabisulfite 97.58 ± 0.53

Sodium citrate dehydrate 95.10 ± 0.39

Carboxymethylcellulose Sodium 96.24 ± 1.44

Hydroxypropyl methylcellulose 98.59 ± 1.33

Table 13: Physical appearance of active–excipients samples before and after storage conditions 40 °C/75%RH for 4 weeks

Phenylephrine hydrochloride and excipients

Initial physical appearance

Physical appearance after 4 weeks of

storage

40 °C/75%RH

Phenylephrine hydrochloride alone Clear solution, no precipitate or residue, no colour change

No change

Benzalkonium chloride and phenylephrine hydrochloride

Clear solution, no precipitate or residue, no colour change

No change

Boric acid and phenylephrine hydrochloride


No change

Sodium carboxy methylcellulose and phenylephrine hydrochloride


No change

EDTA and phenylephrine hydrochloride


No change

Glycerol and phenylephrine hydrochloride


No change

HPMC and phenylephrine hydrochloride


No change

Methyl paraben and phenylephrine hydrochloride


No change

Propyl paraben and phenylephrine hydrochloride


No change

Sodium citrate dihydrate and phenylephrine hydrochloride


No change

Sodium metabisulfite and phenylephrine hydrochloride


No change

The physical appearance of the phenylephrine hydrochloride and excipient blends

can be found in Table 13 above. No colour change and precipitate formation were

observed in any of the 1:1 mixtures after four weeks of storage. There were no

reductions or increase in liquid level after storage, meaning that the plastic container

was not permeable to moisture. The data also proved that the active and excipients

did not interfere or interact with each other.

115

Figure 98: HPLC chromatogram for phenylephrine hydrochloride alone dissolved in mobile phase with retention of 7.82 minutes.

Figure 99: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.

Figure 100: HPLC chromatogram for phenylephrine hydrochloride with sodium citrate dihydrate (1:1) dissolved in mobile phase with a retention time of 7.92 minutes.

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40

45

mAU280nm,4nm (1.00)

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Figure 101: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine

hydrochloride with sodium citrate dihydrate (1:1) in mobile phase. Peak shown in pink and purity

curve in black. Peak purity index = 1.000000; Single point threshold = 0.999896.

Figure 102: HPLC chromatogram for phenylephrine hydrochloride with carboxymethycellulose sodium (1:1) dissolved in mobile phase with a retention time of 7.82 minutes.


hydrochloride with carboxymethycellulose sodium (1:1) in mobile phase. Peak shown in pink and


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117

Figure 104: HPLC chromatogram for phenylephrine hydrochloride with hypromellose (1:1) dissolved in mobile phase with a retention time of 7.81 minutes.

Figure 105: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride with hypromellose (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.

Figure 106: HPLC chromatogram for phenylephrine hydrochloride with glycerol (1:1) dissolved in mobile phase with a retention time of 7.85 minutes.

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80

90

mAU280nm4nm (1.00)

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90

mAU280nm4nm (1.00)

118

Figure 107: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with glycerol (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999989.

Figure 108: HPLC chromatogram for phenylephrine hydrochloride with benzalkonium chloride (1:1)

dissolved in mobile phase with a retention time of 7.88 minutes.

Figure 109: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride with benzalkonium chloride (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999918.

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Figure 110: HPLC chromatogram for phenylephrine hydrochloride with EDTA (1:1) dissolved in mobile phase with a retention time of 7.86 minutes.


hydrochloride with EDTA (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak

purity index = 0.999999; Single point threshold = 0.999991.

Figure 112: HPLC chromatogram for phenylephrine hydrochloride with boric acid (1:1) dissolved in

mobile phase with a retention time of 7.92 minutes.

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250

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120

Figure 113: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with boric acid (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.

Figure 114: HPLC chromatogram for phenylephrine hydrochloride with sodium metabisulfite (1:1)


Figure 115: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with sodium metabisulfite (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999870.

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Figure 116: HPLC chromatogram for phenylephrine hydrochloride with disodium edetate (1:1) dissolved in mobile phase with a retention time of 7.82 minutes.


hydrochloride with disodium edetate (1:1) in mobile phase. Peak shown in pink and purity curve in

black. Peak purity index = 1.000000; Single point threshold = 0.998891.

Figure 118: HPLC chromatogram for phenylephrine hydrochloride with propyl paraben (1:1) dissolved in mobile phase with a retention time of 7.83 minutes.

8.00 8.25 8.50 8.75 min

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122


hydrochloride with propyl paraben (1:1) in mobile phase. Peak shown in pink and purity curve in

black. Peak purity index = 1.000000; Single point threshold = 0.999948.

Figure 120: HPLC chromatogram for phenylephrine hydrochloride with methyl paraben (1:1)


Figure 121: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with methyl paraben (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999982.

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mAUPeak


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123

4.3 Stability study

Stability studies were performed on products I–V at climatic zone II storage

conditions these were 40 °C/75%RH, 25 °C/40%RH and 30 °C/65%RH. The five

products were stored at all three of the above mentioned conditions in triplicate and

tested at zero, one and three months. The most aesthetically pleasing and stable

formulation was chosen based on the phenylephrine hydrochloride left in the

product, pH change and appearance.

The results for storage time zero and one month 40 °C/75%RH (accelerated

condition) were stated in specificity above.

Products I–V stored for 3 months at 30 °C/65%RH: The chromatograms of the

products showed that there was no co-elution and no interference with the API as

shown in Figures 122–131 below. The API left in from products I–V was 72.86%,

96.79%, 75.14%, 98.35% and 61.73% respectively. The difference in mean were

statistically different as p<0.01. The products I and V failed aesthetically as they

turned slightly yellow with no residue, precipitate and no solution loss. The remaining

products remained clear and aesthetically pleasing.

Figure 122: HPLC chromatogram for product I stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

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-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

mAU280nm,4nm (1.00)

124

Figure 123: Peak purity profile calculated using PDA date (from 190–800 nm) for product I stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999950.

Figure 124: HPLC chromatogram for product II stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.85 minutes.

Figure 125: Peak purity profile calculated using PDA date (from 190–800 nm) for product II stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.996473.

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Figure 126: HPLC chromatogram for product III stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes.

Figure 127: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996955.

Figure 128: HPLC chromatogram for product IV stored at 30 °C/65% RH for 3 months dissolved in mobile phase with a retention time of 7.88 minutes.

0.0 2.5 5.0 7.5 10.0 12.5 min

-5.0

-2.5

0.0

2.5

5.0

7.5

mAU280nm,4nm (1.00)

7.75 8.00 8.25 8.50 min

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5.0

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15.0mAU

280nm,4nm (1.00)

126

Figure 129: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.998694.

Figure 130: HPLC chromatogram for product V stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes.

Figure 131: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.994533.

Products I–V stored for 3 months at 25 °C/60%RH: The products showed no co-

elution, no interference, and the API left in products I–V was 82.77%, 96.84%,

7.75 8.00 8.25 8.50 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

mAU280nm,4nm (1.00)

7.75 8.00 8.25 8.50 min

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

mAUPeak


127

86.78%, 97.06%, and 70.88% respectively. There was a statistic difference of

p<0.05. The products III and V failed aesthetically as they turned slightly yellow and

brown respectively, with no residue, precipitate and loss of solution. The remaining

products remained clear and aesthetically pleasing. The figures 132–141 below

show the chromatograms of the products.

Figure 132: HPLC chromatogram for product I stored at 25 °C/60%RH for 3 months dissolved in


Figure 133: Peak purity profile calculated using PDA date (from 190–800 nm) for product I stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.995873.

.

0.0 2.5 5.0 7.5 10.0 12.5 min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5mAU

280nm,4nm (1.00)

7.75 8.00 8.25 8.50 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

mAUPeak


128


Figure 135: Peak purity profile calculated using PDA date (from 190–800 nm) for product II stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.999950.


0.0 2.5 5.0 7.5 10.0 12.5 min

-20

-10

0

10

20

30

40

50

60

70

80

90

100

mAU280nm,4nm (1.00)

7.50 7.75 8.00 8.25 8.50 min

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0

50

100

150

200

250

300

350

400

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

-7.5

-5.0

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

mAU280nm,4nm (1.00)

129


Figure 138: HPLC chromatogram for product IV stored at 25 °C/60%RH for 3 months dissolved in


Figure 139: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996396.

7.75 8.00 8.25 8.50 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

-5.0

-2.5

0.0

2.5

5.0

7.5

mAU280nm,4nm (1.00)

7.75 8.00 8.25 8.50 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

mAUPeak


130



Products I–V stored for 3 months at 40 °C/75%RH: The products showed no co-

elution and no interference. The API left from product I–V were 61.63%, 96.50%,

53.10%, 77.16%, and 54.79% respectively. There was a statistical difference as

p<0.0001. The products I, III and V failed aesthetically as they turned slightly yellow,

dark yellow and brown in colour respectively, with no residue, precipitate and loss of

solution. The remaining products II and IV remained clear and aesthetically pleasing.

The figures 143–152 below show the chromatograms of the products.

0.0 2.5 5.0 7.5 10.0 12.5 min

-5.0

-2.5

0.0

2.5

5.0

7.5

10.0

12.5mAU

280nm,4nm (1.00)

7.75 8.00 8.25 8.50 min

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

mAUPeak


131

Figure 142: HPLC chromatogram for product I stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes.

Figure 143: Peak purity profile calculated using PDA date (from 190 – 800 nm) for product I stored at

40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak



0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

mAU280nm,4nm (1.00)

7.75 8.00 8.25 8.50 min

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

-5.0

-2.5

0.0

2.5

5.0

7.5

mAU280nm,4nm (1.00)

132

Figure 145: Peak purity profile calculated using PDA date (from 190 – 800 nm) for product II stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093.



7.75 8.00 8.25 8.50 min

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min

-5.0

-2.5

0.0

2.5

5.0

7.5

mAU280nm,4nm (1.00)

7.75 8.00 8.25 8.50 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

mAUPeak


133

Figure 148: HPLC chromatogram for product IV stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes.

Figure 149: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at

40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak



0.0 2.5 5.0 7.5 10.0 12.5 min

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

mAU280nm,4nm (1.00)

7.75 8.00 8.25 8.50 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

mAUPeak


0.0 2.5 5.0 7.5 10.0 12.5 min-7

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

mAU280nm,4nm (1.00)

134


Figure 152 below summarises the API remaining in the given storage conditions.

Product II was stable and within the potency range for all storage conditions while

product IV was stable and potent within two storage conditions. The remaining

Products (I, III and V) failed to retain their potency at various conditions. According to

the results obtained, product II and IV were stable and with the most API remaining

in the product. Products II and IV were also the most aesthetically pleasant. This

could be due to the presence of protectors and anti-oxidants such as sodium citrate

dihydrate, EDTA, sodium metabisulfite, boric acid.

Figure 152: A graph showing standard error and phenylephrine hydrochloride left in product I–V after 12 weeks at 30 °C/65%RH, 40 °C/75%RH, 25 °C/60%RH.

7.75 8.00 8.25 8.50 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

mAUPeak


0

20

40

60

80

100

120

1 2 3

Pe

rce

nta

ge

s o

f A

PI

left

in

th

e

pro

du

ct

1 - 30 °C / 65 % RH 2 - 40 °C / 75 % RH 3 - 25 °C / 60 % RH

Phenylephrine hydrochloride left

Product V

Product IV

Product III

Product II

Product I

135

Table 14: Results of one-way ANOVA analysis for products I–V stored at 25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months.

Products °C/%RH

I 25/60

II 25/60

III 25/60

IV 25/60

V 25/60

I 30/65

II 30/65

III 30/65

IV 30/65

V 30/65

I 40/75

II 40/75

III 40/75

IV 40/75

V 40/75

I 25/60 **** **** **** **** **** **** **** **** **** **** **** **** **** ****

II 25/60 **** **** ns **** **** ns **** ** **** **** ns **** **** ****

III 25/60 **** **** **** **** **** **** **** **** **** **** **** **** **** ****

IV 25/60 **** ns **** **** **** ns **** * **** **** ns **** **** ****

V 25/60 **** **** **** **** *** **** **** **** **** **** **** **** **** ****

I 30/65 **** **** **** **** *** **** **** **** **** **** **** **** **** ****

II 30/65 **** ns **** **** **** **** **** ** **** **** ns **** **** ****

III 30/65 **** **** **** **** **** **** **** **** **** **** **** **** **** ****

IV 30/65 **** ** **** **** **** **** ** **** **** **** *** **** **** ****

V 30/65 **** **** **** **** **** **** **** **** **** ns **** **** **** ****

I 40/75 **** **** **** **** **** **** **** **** **** ns **** **** **** ****

II 40/75 **** ns **** **** **** **** **** **** *** **** **** **** **** ****

III 40/75 **** **** **** **** **** **** **** **** **** **** **** **** **** **

IV 40/75 **** **** **** **** **** **** **** **** **** **** **** **** **** ****

V 40/75 **** **** **** **** **** **** **** **** **** **** **** **** ** ****

ns - Not Significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001

Above is Table 14 which shows results of statistical comparisons of all the products.

The quantity of phenylephrine hydrochloride in product II did not change significantly

when the remaining amounts of API for the various storage conditions were

compared. The quantity of API present for the other products changed significantly,

this means that product II was stable at all three storage conditions.

Summarised below in tables 15 and 16 are the changes in pH for products I–V.

Table 15 below shows pH change in products I–V stored at 40 °C/75%RH for one

month, Table 16 below shows pH changes in products I–V stored at various storage

conditions after three months of storage. Changes were observed for some products,

as they deviated from the pH of four they were initially formulated at. This could be

due to oxidation or reduction reactions of the API or excipients; it should be noted

that phenylephrine hydrochloride is stable in the pH range of 3.5–8 and none of the

products exceeded this limits even though changes were noted (Giahi et al., 2009).

Over time products I, III and V became more basic, product II and IV were stable and

remained close to their original pH for all storage conditions. According to the pH

results obtained, product II and product IV showed favourable stability.

136

Table 15: Changes in pH of phenylephrine hydrochloride and products I–V before and after storage conditions 40 °C/75%RH for 1 month.

Item Temperature reading (°C)

pH before Temperature reading (°C)

pH after 1 month

Phenylephrine Hydrochloride

20 4.0 20 3.59 ± 0.89

Product I 20 4.0 20 6.25 ± 0.10

Product II 20 4.0 20 3.95 ± 0.87

Product III 20 4.0 20 5.82 ± 0.45

Product IV 20 4.0 20 3.99 ± 0.38

Product V 20 4.0 20 6.10 ± 0.25

Bold print indicates a change in pH after 4 weeks of storage

Table 16: Changes in pH for products I–V before and after varying storage conditions for 3 months

Item Temperature reading (°C)

before storage

pH before storage

Temperature reading (°C) after storage

40 °C/75%RH

30 °C/65%RH

25 °C/60%RH

Product I 20 4.0 20 5.9 ± 0.16 6.12 ± 0.16

6.18 ± 0.16

Product II 20 4.0 20 3.63 ± 0.57 3.88 ± 0.39

3.90 ± 0.82

Product III 20 4.0 20 4.67 ± 0.65 4.87 ± 0.31

6.03 ± 0.41

Product IV 20 4.0 20 3.63 ± 0.27 3.63 ± 0.68

3.96 ± 0.25

Product V 20 4.0 20 3.23 ± 0.47 4.2 ± 1.4 5.68 ± 0.17

Bold print indicates a change in pH after 3 months of storage

Statistical differences were determined using the one-way ANOVA analysis. All pH

results were significantly different from each other as p<0.0001; similarities were

summarized in Table 17 below.

Table 17: Results of one-way ANOVA analysis for similarities in pH of products II 25 °C/60%RH, 40 °C/75%RH and IV 25 °C/60%RH after 3 months.

°C/%RH

I 25/60

II 25/60

III 25/60

IV 25/60

V 25/60

I 30/65

II 30/65

III 30/65

IV 30/65

V 30/65

I 40/75

II 40/75

III 40/75

IV 40/75

V 40/75

IV

25/60

ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

II

40/75

**** **** **** **** **** **** **** **** ns **** **** **** **** **** ****

II

25/60

**** **** **** ns **** **** ns **** **** **** **** **** **** **** ****

NS - Not Significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001

137

Table 18: Physical appearance of products I–V before and after varying storage conditions for 3 months.

Item Initial

Physical appearance

Physical appearance After 3 months of storage

40 °C/75%RH 30 °C/65%RH 25 °C/60%RH

Product I Clear solution, no precipitate or residue, no colour change

Clear solution, yellow in colour, no residue, no precipitate, no loss of solution

Slightly yellow, clear, no residue, no precipitate, no loss of solution

No change in initial appearance, no loss of solution

Product II Clear solution, no precipitate or residue, no colour change


Clear, no residue, no precipitate, no loss of solution


Product III Clear solution, no precipitate or residue, no colour change

Clear solution, tinge yellow, no residue, no precipitate, no loss of solution


Slight yellow, no precipitate formed

Product IV Clear solution, no precipitate or residue, no colour change




Product V Clear solution, no precipitate or residue, no colour change

Dark brown, no precipitate formed, presence of residue, no loss of solution

Brown, no residue, no precipitate, no loss of solution

Tinge brown, no precipitate formed, no loss of solution, no residue

Bold print indicates a change after 3 months of storage

Changes in physical appearance were judged visually. Aesthetically pleasing

products were noted and above in Table 18 is a summary of what they looked like

after three months, products II and IV were the only ones that did not have physical

changes.

4.4 Determination of yield point and viscosity of products

The viscosity of a solution is a particluarly important parameter, especially during

production as continous quality control is essential in order to achive consitently high

quality despite the immense production volume. The graphs seen in figures 153–158

below show the flow curves of products I–V and a marketed product Prefrin® at

different storage conditions. With the aid of Herschel/Bulkley formula, yield points

were calculated and measured by Anton Paar rheometer (RheoPlusTM).

Good viscosity properties of the VMA is important in this experiment, as viscosity

shows the flow movement, while yield points corresponds directly to the elastic

properties of the formulation. The product must flow out of the plastic eye drop bottle

but should be viscous enough for accurate drop dosing.

138

Figure 153: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 30 °C/65% RH.

Figures 153–158 show variance in viscosity between the different viscosity modifiers

(glycerol, hydroxy propyl methylcellulose and sodium carboxy methyl cellulose). At

time zero, Prefrin® and product I–V display slightly similar flow. After three months

glycerol had a marked increase in its viscosity, this is seen in the figures 154, 156

and 158 below. Other viscosity modifiers show slight changes.

Figure 154: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage condition 30 °C/65% RH.

0

2

4

6

8

10

12

14

16

18

20

22

24

26

30

Pa

10-3

10-2

10-1

100

Pa·s

0 100 200 300 400 500 600 7001/s

Shear Rate .

TAU

Anton Paar GmbH

hpmc product 5 30/65 3 t0 2

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

glycerol product 3 30/65 3 t0 2

CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity

scmc product 4 30/65 3 t0 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

Prefrin 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

0

2

4

6

8

10

12

14

16

18

20

22

24

26

30

Pa

10-3

10-2

10-1

100

Pa·s

0 100 200 300 400 500 600 7001/s

Shear Rate .

TAU

Anton Paar GmbH

glycerol product 330/65 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

hpmc product 2 30/65 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity

scmc product 4 30/65 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

Prefrin 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

139

Figure 155: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 40 °C/75% RH.


0

2

4

6

8

10

12

14

16

18

20

22

24

26

30

Pa

10-3

10-2

10-1

100

Pa·s

0 100 200 300 400 500 600 7001/s

Shear Rate .

TAU

Anton Paar GmbH


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity

scmc product 4 40/75 3 t0 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

Prefrin 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

0

2

4

6

8

10

12

14

16

18

20

22

24

26

30

Pa

10-3

10-2

10-1

100

Pa·s

0 100 200 300 400 500 600 7001/s

Shear Rate .

TAU

Anton Paar GmbH


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity

glycerol product 3 40/75 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

Prefrin 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

140

Figure 157: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 25 °C/60%RH.


0

2

4

6

8

10

12

14

16

18

20

22

24

26

30

Pa

10-3

10-2

10-1

100

Pa·s

0 100 200 300 400 500 600 7001/s

Shear Rate .

TAU

Anton Paar GmbH


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity

scmcproduct 4 25/60 3 t0 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

Prefrin 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

0

2

4

6

8

10

12

14

16

18

20

22

24

26

30

Pa

10-3

10-2

10-1

100

Pa·s

0 100 200 300 400 500 600 7001/s

Shear Rate .

TAU

Anton Paar GmbH

glycerol product 3 25/60 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity


CC27-SN25781; d=0 mm

Shear Stress

Viscosity

Prefrin 3 1

CC27-SN25781; d=0 mm

Shear Stress

Viscosity

141

Prefrin® was choosen as the product against which others would be measured

because polvinyl alcohol was used as its VMA, while phenylephrine hydrochloride as

its API. This made it a good chioce for comparison as it had a VMA and a similar

API. It proved to be not significantly as different to the rest as seen in figures 154–

159 as p>0.05. Products I–V did not lose water from the containers during the

stability study, thus the change in viscosity cannot be attributed to loss of volume.

Figure 159: Graph showing viscosity of products I–V stored in a stability chamber of 40 °C/75%RH tested at time zero (T0), three months later (T1) and compared to an original marketed product Prefrin®.

Figure 160: Graph showing viscosity of products I–V stored in a stability chamber of 25 °C/60%RH tested at time zero (T0), 3 months later (T1) and compared to an original marketed product Prefrin®.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Product I Product II Product III Product IV Product V Prefrin®

Vis

cosi

ty (

mP

a.s)

T1 40/75

T0 40/75

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


Vis

cosi

ty (

mP

a.s)

t1 25/60

T0 25/60

142

Figure 161: Graph showing viscosity of products I–V stored in a stability chamber of 30 °C/65%RH tested at time zero (T0), 3 months later (T1) and compared to an original marketed product Prefrin®.

Using the above graphs in figures 159–161 product II showed stability and good

rheology properties. The higher the value is to 1 mPa.s, the more viscous a product

was.

Statistically no difference was found among most products manufactured and the

marketed product. However, product III had significant differences (p<0.05) in

viscosity at various storage conditions and time frames as seen in Table 19 below.

Product III had a significantly higher viscosity than the other products after the 3

months stability period. The products with “ns” showed that the formulations were the

same in mean flow and yield points.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


Vis

cosi

ty (

mP

a.s)

T1 30/65

T0 30/65

143

Table 19: Results of one-way ANOVA analysis for viscosity of products I–V stored at 25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months. The values shown indicate differences in p-values and significance in differences of mean was defined as p < 0.05.

mPa.s

PI 25/60

PII 25/60

PIII 25/60

PIV 25/60

PV 25/60

PI 30/65

PII 30/65

PIII 30/65

PIV 30/65

V 30/65

I 40/75

II 40/75

III 40/75

IV 40/75

V 40/75

Prefrin

®

PI 25/60


PII 25/60


PIII 25/60

ns ns ns ns ns ns ns ns ns ns ns ns ns ns **

PIV 25/60


PV 25/60


PI 30/65


PII 30/65


PIII 30/65

ns ns ns ns ** ns ns * ns *** * ns ** * ****

PIV 30/65


PV 30/65


PI 40/75


PII 40/75


PIII 40/75

ns ns ns ns ns ns ns ns ns ns ns ns ns ns **

PIV 40/75


PV 40/75


Prefrin

®


ns - Not Significant, *= p < 0.05, ** = p < 0.01, ***=p < 0.001, **** = p < 0.0001

144

4.5 Effectiveness of the ophthalmic solution preservatives

Ophthalmic drops are sterile formulations which are usually packed in multi-dose

containers. Microbial contamination from multiple uses may lead to product

degradation or result in ocular infection (Sutton et al., 2002). Protection of these

multiple dose products against microbial contamination is usually achieved by

addition of a suitable preservative system. The antimicrobial effectiveness test is

designed to provide a laboratory test that gauges the level of antimicrobial activity by

a pharmaceutical product and to evaluate how well a product withstands microbial

contamination while being used (Nostro et al., 2004 and Sutton et al., 2002).

The antimicrobial preservative efficacy of the eye-drops challenged with E. coli, S.

aureus, P. aeruginosa, and C. albicans showed varied results as seen in Table 20

below. The control eye-drop formulation failed in its preservative efficacy as it lacked

any form of preservative. After six hours all formulations containing preservatives

showed reduction in the initial microbial count. Four of the eye-drops eradicated the

inoculated microorganisms by more than 3 logs in 24 hours, except product IV,

which remained as log 2.

The number of P. aeruginosa, C. albicans, E. coli and S. aureus in product IV

decreased gradually while the remaining products decreased by over two to three

logs. This means that the EDTA, boric acid, sodium metabisulfite and sodium citrate

dihydrate acted synergistically to prevent increase in microbial count of other

products. For the stated excipients, EDTA had antioxidant and antimicrobial

properties; boric acid had antiseptic properties while sodium metabisulfite and

sodium citrate had small preservative powers. Products III and V, which contained

methylparaben and propylparaben showed the most decrease in microbial load.

After 14 days, all the eye-drops had varied log reduction against all the challenging

organisms which were all within acceptable limits. In all cases the number of fungi

after 7, 14 and 28 days were acceptable as they were reduced by 3 and 4 logs which

surpassed the requisite of fungi count.

145

Table 20: Antimicrobial preservative efficacy of the eye-drop products I–V challenged with E. coli, S.aureus, P. aeruginosa, C.albicans. Microorganism Eye-drop Sampling time/Log reduction

E. coli ATCC 38218

0 hour

6 hours 24 hours

7 days 14 days 28 days

Product I 2.9 x 10

6 3 2 - - NR

Product II 2.7 x 10

6

3 3 - - NR

Product III 2.7 x 10

6

4 1 - - NR

Product IV 2.9 x 10

6

2 2 - - NR

Product V 2.9 x 10

6

4 2 - - NR

S.aureus ATCC 43300

Product I 1.1 x 10

6 3 2 - - NR

Product II 1.1 x 10

6

3 3 - - NR


6

3 2 - - NR

Product IV 2.0 x 10

6

2 2 - - NR

Product V 1.1 x 10

6

4 2 - - NR

P. aeruginosa ATCC 27853

Product I 3.8 x 10

6 3 2 - - NR

Product II 3.8 x 10

6

3 3 - - NR


6

4 1 - - NR

Product IV 3.5 x 10

6

2 2 - - NR

Product V 3.5 x 10

6

4 2 - - NR

C. albicans ATCC 66027

Product I 2.1 x 10

6 - - 3 2 NI

Product II 2.0 x 10

6

- - 3 - NI


6

- - 4 2 NI

Product IV 2.1 x 10

6

- - 3 2 NI

Product V 2.0 x 10

6

- - 4 - NI

NR – no recovery of microbial load, NI – no increase of fungi growth.

Eye drops are classified under category 1 in the USP (2007). The criteria for

category one under the USP (2007) goes as thus, “bacteria, not less than 1.0 log

reduction from the initial calculated count at seven days, not less than 3.0 log

146

reduction from the initial count at 14 days, and no increase from 14 days’ count at 28

days”. The fungus that was specified showed no increase from initial calculated

count at 7, 14 and 28 days. No increase was defined as not more than 0.5 log10 unit

higher than the previous value measured. The requirements for antimicrobial

effectiveness of all the products were met.

147

5. CONCLUSION AND RECOMMENDATIONS

The HPLC method used in the present study permitted rapid and precise

determination of phenylephrine hydrochloride and complied with the requirements for

specificity, linearity, accuracy, precision and range. The method was accepted as

valid, and was therefore deemed suitable for quantitative assay of phenylephrine

hydrochloride in the finished products during initial formulation analysis and for the

ensuing stability studies.

Active-excipient compatibility studies were carried out in order to determine which

excipients would have a potential for reacting unfavourably with phenylephrine

hydrochloride. From the above results, HPLC showed it could be used as a method

of analysis for active-excipient mixtures of phenylephrine hydrochloride products.

Excipients analyzed in the active-excipient compatibility study, demonstrated

compatibility with phenylephrine hydrochloride as no interfering peaks or co-eluting

peaks were found. If such occurred it would be regarded as an impurity to the API. A

1:1 ratio of active-excipient mixture was used; while, in the actual formulation, the

ratio of excipients to phenylephrine hydrochloride was small, minimizing the potential

for interaction. The chromatograms showed no interference from impurities or

degradation of product. The results showed compatibility between phenylephrine

hydrochloride and its excipients.

The products were analyzed in order to determine which formulations produced a

physically and chemically stable product after stability testing. The API remaining

after the three storage conditions differed among the products I–V; product II and IV

stayed within accepted concentrations of greater than 90% among the three different

storage conditions, however, product II was the only one that showed no statistical

difference during storage. The remaining products failed to achieve the same

concentrations. Product III and V fared better in the 25 °C/60%RH but the

concentrations were still below the accepted range. The better stability could be due

to the fact that products II and IV had excipients with antioxidant and buffering

properties, sodium metabisulfite and sodium citrate dihydrate for product II, EDTA

and boric acid for product IV. Product II was stable and within potency range in all

148

storage condition whereas product IV was stable and potent in two storage

conditions namely 25 °C/60%RH and 30 °C/65%RH. The remaining Products (I, III,

V) failed to retain their potency at most storage conditions.

Colour and pH changes were observed mostly with no loss or increase of solution.

Observations with regard to no loss of solution meant that the container was suitable

for the formulations and it was impervious to moisture. Further observations were

that products III and V showed degradation of phenylephrine hydrochloride. This was

clearly depicted as phenylephrine hydrochloride within the product formulation of III

and V was brown or reddish in colour. This meant the phenylephrine hydrochloride

had oxidized fully over the period of three months in which the products were

stressed in variable storage conditions. It shows that phenylephrine hydrochloride

formulations which are in solutions require antioxidants or protective agents and

buffering agents to reduce its quick breakdown, product II and IV had both.

The pH of the products varied from 3.5 to 8, all within the stable range of

phenylephrine hydrochloride. From the assay results of HPLC, phenylephrine

hydrochloride loses its potency when reacted with hydrogen peroxide and sodium

hydroxide. As the pH of the products increases there is a decrease in the

concentration of phenylephrine hydrochloride. Product II an IV stayed within the pH

of 3 meaning acidity kept PE from degrading. Any future research could focus on

optimizing buffers and chemicals that protect phenylephrine from degrading.

Rheological tests conducted displayed notable findings especially with glycerol

included in product III. Prefrin® (polyvinyl alcohol) was used as standard to which the

products were compared. In all, three viscosity-modifying agents were used for

products I–V and they were HPMC, SCMC and glycerol. Figures 154–158 indicate

that Prefrin® did not display superior viscosity when compared to products I–V.

Glycerol increased in its viscosity over time, the reason is still unknown as it is said

to be a stable excipient. A study should be conducted on the glycerol containing

product to determine why there is an increase in viscosity over time. Viscosity

modifying agents, HPMC and SCMC are similar in function but physicochemically

dissimilar (Kibbe, 2006). The viscosity of SCMC loses 25% of its original strength

when autoclaved while HPMC only gets denatured at extremely high temperatures

149

(Parsons, 2006). Generally, increased temperature (autoclaving and storage

conditions) decreases the viscosity of the stated three viscosity modifying agents,

the important function needed is the ability to return back to their original state. With

this being noted, product IV recovered its viscosity during rheological tests of time

zero and three months later while product II also recovered but not completely.

Product I failed to recover at the storage condition 40 °C/75%RH. This could mean

that SCMC and HPMC could have lost some of their viscosity due to cellulose gum

depolymerization. This in turn adds to the reason why it in many cases needs

additives and preservatives. Another fact that was observed from literature written by

Junyan and colleagues (2009) was that EDTA protects cellulose from loss of

viscosity; this could have enabled SCMC and HPMC in achieving their return to initial

viscosity. Product II, III and IV showed best performances with regard to their flow.

According to the results obtained, product II and IV were favourable. Any future

research could focus on optimizing favourable viscosity-modifying range for

hydroxylpropyl methylcellulose and carboxy methylcellulose sodium. The optimal

value for phenylephrine hydrochloride protectors such as sodium citrate dihydrate,

EDTA, sodium metabisulfite, boric acid should be studied.

Another aspect from the objectives was to find whether the products were provided

with preservatives which were effective to inhibit microbial growth. The result showed

a positive answer. All products reduced the microbial load that they were inoculated

with. Also they passed the compendia requisite for category 1 products (eye drops).

Product I, II, III and V were successful in fulfilling the compendial requirement of

preservative efficacy.

Overall, product II was selected as the best among the five products. It would be

beneficial to approach its development on a more intensive and rigorous scale as to

determine if it would be passed as a generic product for the market at large.

Some limitations of the study were due to reduced sample size. It could have been

increased to ensure a representative distribution in differences and possibly

similarities among products. An increase in formulations samples could lead to

150

increased chances of a more favourable and stable solution. This allows for wider

research to find the best fit ingredients to be mixed together to provide the desired

solution. Reliable reports on phenylephrine hydrochloride in solutions would enhance

quicker decisions in stabilization, as those available are patented and require

substantial amount of money to acquire and use. Extra methods could have been

applied in testing the available samples. An example is the full factorial experimental

design, this design makes it possible to examine all possible combinations of

ingredient variant are served with equal probability.

151

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tropicamide (paremyd®) and 0.5% tropicamide combined with 2.5% phenylephrine.

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Research, vol. 82, Issue 5, pp 885–898.

176

APPENDIX A

CONCEPT ARTICLE

The following manuscript is intended to be submitted for publication.

Use and validation of high performance liquid chromatography for

phenylephrine hydrochloride estimation

Chinedum Okafor a, Mbali Keele a*, Gareth Kilianb, Matthew Worthingtonc

a Department of Pharmacy, PO Box 77000, Nelson Mandela Metropolitan University,

Port Elizabeth, 6031

b University of Western Cape

c Aspen Pharmacare, 7 Fairclough Road, Korsten, Port Elizabeth, 6014

* E-mail: [email protected]

Abstract:

Simple, accurate and reproducible high performance liquid chromatography (HPLC)

method was validated for the estimation of phenylephrine hydrochloride in

pharmaceutical eye drop formulations. Phenylephrine hydrochloride (PE) was

assayed using HPLC at concentration range of 0.0125 to 0.15 mg/ml. Linearity, y =

8541.1 x + 438.55 was achieved as the range were directly proportional to the

concentration of phenylephrine hydrochloride within a given range (r2 = 0.9999). The

method was tested and validated for various parameters according to the ICH

(International Conference on Harmonization) guidelines. The detection and

quantification limits were found to be 12.3 and 41 μg/ml respectively. The proposed

method was successfully applied for the determination of phenylephrine

hydrochloride in pharmaceutical eye drop formulations. The results demonstrated

that the procedure is accurate, precise and reproducible, while being simple, cheap

177

and less time consuming, and hence can be suitably applied for the estimation of

phenylephrine hydrochloride in eye drops.

Key words: phenylephrine hydrochloride; HPLC; formulations; eye drops

Introduction

Phenylephrine hydrochloride (PE) is a potent adrenergic agent and β–receptor

sympathomimetic drug, used in its optically active form (Pandey et al., 2003; Pandey

et al., 2006). As an α1-adrenergic receptor agonist it is used primarily as a

decongestant, for uveitis and as an agent to dilate the pupil (Lang, 1995).

The drainage of phenylephrine hydrochloride into the nasal mucosa could result in

systemic absorption of this agent and produce many unwanted systemic side effects

including tachycardia, hypertension, and headache (Bartlett and Jaanus, 2008).

Also, the eye drop solutions were either blinked out or only a small portion of the

drug reached its site of action. The use of viscosity modifying agents is included in

solutions with the aim of obtaining thickening effects. However, these components

may have other effects, whether independently or as a consequence of interactions

with other components, these effects being mostly due to electrostatic, steric,

electrosteric, or depletion mechanisms (Duro et al., 1999).

Various methods have been reported in the literature for the analysis of

phenylephrine hydrochloride including spectrophotometer (Collado et al., 2000; Erk,

2000; Solich et al., 2000; Shama, 2002; Knochen & Giglio 2004), spectrophotometry

with chromogenic reagent (Ahmed and Amin, 2007), fluorometry (Martin et al.,

1993), High-performance liquid chromatography (Marin et al., 2002; Olmo et al.,

2005; Galmier et al., 2000) spectro-fluorimetric and derivative spectrophotometric

methods (Sabry et al., 2000), have also been reported for the determination of

phenylephrine hydrochloride.

The aim of the study was is to use and validate a HPLC analytical method for the

estimation of phenylephrine hydrochloride in pure form and in pharmaceutical eye

178

drop formulations. The method was validated as per ICH (International Conference

on Harmonization) guidelines and MCC requirements.

Materials and methods

Materials

Phenylephrine hydrochloride, sodium citrate dihydrate, boric acid, disodium

edentate, octane-1-sulfonic acid sodium salt, sodium metabisulphite, and

benzalkonium chloride were kindly donated by Aspen Pharmacare (Port Elizabeth,

SA). Water for chromatography was produced by Ultra Clear TWF/El-Ion® system

which has been pre-treated and made ultrapure (reverse osmosis) (Separations,

Johannesburg, SA). HPLC grade methanol and octane-1-sulfonic acid sodium salt

was obtained from Sigma-Aldrich (Pty) Ltd (Kempton Park, SA). Analytical / technical

grades of sodium hydroxide pellets, carboxymethylcellulose sodium,

hydroxymethylcellulose, glycerol, methanol hydrochloride acid solution 33%,

pyrophosphoric acid (phosphoric acid) was obtained from Merck Laboratory Supplies

(Pty) Ltd (Midrand, SA) and hydrogen peroxide 30% were sourced from Saarchem

(Pty) Ltd (Johannesburg, SA).

Instruments

The HPLC system consisted of a complete FPLC Shimadzu® HPLC system which

has a SPD-M20A Prominence diode array detector, SIL-20A Prominence auto-

sampler, DGU-20A5 Prominence degasser, LC-20AB Prominence liquid

chromatography and CTO-10AS vp Prominence column oven ( Shimadzu, Tokyo,

Japan). Column was a reverse phase Phenomenex® Luna C18 (2) column 250 mm

× 4.60 mm, 5 μm particle size (Separations, Johannesburg, SA).

179

Calibrations

1.1g of octane-1-sulfonic acid sodium salt was dissolved in one litre mixture of

methanol and water (1:1) and the pH was adjusted to 3.0 with pyrophosphoric acid.

The resulting solution was mixed, degassed by ultrasonication (Ultrasonic LC 130,

Labotec, Germany) and vacuum filtered through a 0.45 μm Millipore filter (Millipore

Corporation, Bedford, Massachusetts, USA) prior to use. Dilution solvent was

prepared as a mixture of HPLC grade methanol and water (1:1) and adjusted to a pH

of 3. The stock solution was prepared by accurately weighing 200 mg of

phenylephrine hydrochloride material into a 100 ml volumetric flask, dissolving it and

making up to volume with dilution solvent. Calibration standards containing 0.0125,

0.025, 0.05, 0.075, 0.1 and 0.15 mg / ml were prepared by making appropriate

solvent dilutions of the working stock solution. Each calibration standard was filtered

through a 0.45 μm Millex® syringe driven filter unit prior to injection.

Sample preparation

Eye drop formulations had PE concentration of 100 mg / ml. The eye drop

formulations were filtered and 0.3 ml was dissolved into 10ml of dilution solvent to

get a final concentration of 0.03 mg / ml. The samples were analyzed using the

following analytical method.

Methods

The samples were analyzed using the following analytical method:

Linearity

Linearity of an analytical procedure is its ability, within a given range, to obtain test

results that are directly proportional to the concentration of analyte in the sample

(ICH Harmonized Tripartite Guideline Q2A, 2005). A calibration curve was prepared

and linearity demonstrated over a phenylephrine hydrochloride concentration range.

A stock solution was prepared having a known concentration of 2 mg / ml (defined as

100%) and dilution of the stock solution to final concentrations of 0.0125 to 0.15 mg /

ml were prepared with reverse osmosis ( RO) water and filtered through 23 mm 0.45

180

µm PVDF syringe filters (Millex-HV, Millipore, Billerica, USA). Each of the standards

was assayed in triplicate. The calibration curve was constructed by plotting the peak

areas of phenylephrine hydrochloride versus the respective phenylephrine

hydrochloride concentrations and a linear regression trend line was fitted to the plot

on Microsoft Excel® 2007, Microsoft Corporation.

Accuracy and precision

Accuracy and precision were determined by replicate injection (n=6) of three

phenylephrine hydrochloride solutions, at the upper, middle, and lower limits of the

concentration range studied. The concentration ranges were 0.0095 lower limit,

0.054 middle limit and 0.138 upper limit (mg/ml). The theoretical concentrations were

calculated from the linear regression curve, and compared to the actual

concentrations obtained. The actual mean concentration and standard deviation

were calculated at each theoretical concentration. The mean concentrations and

percentage recovery of phenylephrine hydrochloride obtained for the replicate

injections were a measure of the accuracy of the method, whilst the relative standard

deviations at any one concentration provided a measure of precision. The

requirement for accuracy is that the percentage recovery of phenylephrine

hydrochloride for each solution prepared must be within the 98.00 to 102.00% limit.

The requirement for precision is that the relative standard deviations at any one

concentration must be less than or equal to 2.00%.

Limit of detection and limit of quantification

Standard solutions of decreasing concentration were produced by successive

dilution of the lowest calibration standard and the resulting solutions were injected in

triplet. 0.01 ml of the least calibration standard concentration was diluted 10 times.

Specificity

All five products and the phenylephrine hydrochloride were subjected to the following

stress conditions after which they were manipulated and analysed:

0.2 M NaOH for 30 minutes (reflux system);

0.2 M HCl for 30 minutes (reflux system);

181

0.2 M H2O2 for 30 minutes (reflux system);

UV lights (17 hours inside a stability chamber);

100 °C (24 hours inside a stability chamber);

65 °C (1 month inside a stability chamber);

40 °C/75%RH (1 month inside a stability chamber);

Unstressed batch of phenylephrine hydrochloride and Products I–V

A mass of 10 mg of unstressed phenylephrine hydrochloride was dissolved in 100 ml

of dilution solvent and analysed using the HPLC method being validated. A volume

of 0.3 ml of unstressed Product I–V was dissolved in 10 ml dilution solvent and

analysed using the HPLC method being validated. The same method was applied for

the samples (phenylephrine hydrochloride and products I–V) stressed within the

stability chamber (UV lights, 65 °C, 100 °C, 40 °C/75%RH). Phenylephrine

hydrochloride (10 mg) was diluted to 100ml 0.2 M HCl, 5 ml was diluted to 25 ml 0.2

HCl and refluxed for 30 minutes; the same manipulation was applied when using

NaOH and H2O2. 0.5ml of product I–V was diluted to 100 ml 0.2 M HCl and refluxed

for 30 minutes, 10 ml was diluted to 100ml dilution solvent, and analysed using the

HPLC method being validated. The same manipulation was applied when using

NaOH and H2O2. Samples (phenylephrine hydrochloride and Products I–V) were

stressed in a stability chamber (Binder, SA) which emitted both UV and visible light

through a window glass filter (type 2) and did conform to the requirements of the ICH

guidelines. The stability chamber had an irradiance level of 318 watts / m2 in order to

expose the samples to an overall illumination of not less than 1.2 million lux hours

and an integrated near UV energy of not less than 200 watt hours / m2 all according

to ICH Harmonized Tripartite Guideline Q1B, 2005.

Results and discussion

Linearity

Linearity indicates that the method a calibration curve was constructed by plotting

the area of the phenylephrine hydrochloride peak versus phenylephrine

hydrochloride concentration. The figure below shows linearity over the concentration

182

range. The linear regression equation for the concentration range of 0.0125 to 0.15

mg/ml was y = 8541.1x + 438.55, with a correlation coefficient, R2, equal to 0.9999.

The requirements for linearity were attained, as the correlation coefficient of the

regression line was greater than 0.999 and the percentage relative standard

deviations for the phenylephrine hydrochloride peak areas of multiple injections were

all less than 1.5 %. The y-intercept was found to be 2 > z > -2. This means that the

results achieved are directly proportional to the concentration of phenylephrine

hydrochloride within a given range.

Figure 1: Graph showing a mean peak area versus concentration of replicate samples of phenylephrine hydrochloride standards. Linear regression equation: y = 8541.1 x + 438.55, R2 = 0.9999.

0

200000

400000

600000

800000

1000000

1200000

1400000

0 50 100 150 200

pe

ak

are

a

concentration (mg/ml)

183

Accuracy

Accuracy was within acceptable range as noted in Table 1. The percentage relative

standard deviations calculated for the samples at the lower, middle and upper limits

of the concentration range were all below 0.5%. The recovery percentage for the

samples was between the limits of 99.00 to 100.10%. This means by applying the

analytical method of a known purity concentration (linearity) and comparing the

results of a second, well characterised method, the difference should not be greater

than 0.5%.

Precision

Precision was within acceptable range as seen above in Table 2. The percentage

relative standard deviations calculated for the samples at the lower, middle and

upper limits of the concentration range were all below 0.5%. The recovery

percentage for the samples was between the limits of 99.00 to 100.00%. The

precision method had the degree of agreement among individual test results when

the method was applied repeatedly to the three concentrations chosen of

phenylephrine hydrochloride. As above it is expressed in RSD, showing that it can

be reproduced or repeated under normal operating conditions.

Limit of detection (LOD)

The LOD was found to be 12.3 μg/ml. The amount stated is the lowest amount of

phenylephrine hydrochloride in a sample that can be detected. The LOD is the

lowest concentration for which the relative standard deviation of multiple injections is

less than 5.0%. By convention, the LOD value is taken as 0.3 times the LOQ

(Armbruster & Pry, 2008).

184

Limit of quantification (LOQ)

The LOQ was found to be 41 μg/ml. The amount shows the lowest amount of analyte

in a sample that can be determined with acceptable precision and accuracy. LOQ =

3.33 LOD (Thomsen et al., 2003).

Specificity

Specificity of a chromatographic method is the ability of the method to accurately

measure the analyte response in the presence of all potential sample components.

Specificity is useful to show that an analyte response cannot be attributed to more

than one component (Rozet et al., 2011). The chromatograms were examined for the

presence of compounds, metabolites, impurities, degradants that may interfere or

partly co-elute with the phenylephrine hydrochloride peak.

The results are shown below:

1. Mobile phase chromatogram showed no interference. The mobile phase

produced a chromatogram which had a steady baseline and no ghost peaks

as seen in Figure 2.

2. Phenylephrine hydrochloride peak observed, it shows no interference from

contaminants or impurities. It eluted (had a retention time) of 7.8 minutes. The

peak was symmetrical not fronting or tailing.

3. All products that were stressed using UV light, heat, humidity, HCl and NaOH,

did not produce compounds which interfered or co-eluted with phenylephrine

hydrochloride. This means the excipients did not react with the phenylephrine

hydrochloride or any degradants co-eluting with the phenylephrine

hydrochloride. The products changed from clear solution to a brownish colour

during base stress refluxing, while a pale yellow–lime green colour was

observed during peroxide stress refluxing. No change in colour was observed

during UV stress testing.

4. The mobile phase was different from the extraction as a result a constant

peak is noted between 2.5 and 3.5 minutes as observed in figures 2–6. It

takes that amount of time for the injected sample to reach the column. This

peak is constituent for all chromatograms.

Peak purities were observed in all samples of stressed and unstressed

phenylephrine hydrochloride and finished product solutions. Phenylephrine

185

hydrochloride showed loss to the combination of heat and humidity. It was, however,

potent against heat alone. There was decomposition by acid, peroxide and base.

Phenylephrine hydrochloride was stable to UV light.

Conclusion

High performance liquid chromatography method was used successfully for

phenylephrine hydrochloride determinations in eye drop formulations. The analytical

method was simple, sensitive, rapid and specific and it can be conveniently used for

analysis and quality control of phenylephrine hydrochloride in eye drop formulations.

The method was suitable to determine concentrations in the range 0.0125–0.15

mg/ml precisely and accurately. The limits of detection and quantification were

detected as 12.5 and 41 μg/ml respectively.

Reference

Ahmed, I.S. Amin, A.S. 2007. Spectrophotometric Microdetermination of

Phenylephrine Hydrochloride in Pure and in Pharmaceutical Formulations Using

Haemotoxylin. Journal of Molecular Liquids, vol. 130, Issues 1–3, pp 84–87.

Armbruster, D.A. & Pry, T. 2008. Limit of Blank, Limit of Detection and Limit of

Quantitation. Clinical Biochemical Review, vol. 29, Issue 1, pp 49–52.

Barlett, J. D. & Jaanus S. D. 2008. Clinical Ocular Pharmacology. 5th edition.

Churchill Livingstone: Elsevier Health Sciences, pp 1–50.

Collado, M. S. Mantovani, V. E. Goicoechea, H. C. Olivieri, A. C. 2000.Simultaneous

spectrophotometric-multivariate calibration determination of several components of

opthhalmic solutions: phenylephrine, chloramphenical, antipyrine, methylparaben

and thimerasal, Talanta, Issue 52, pp 909–920.

Duro, R. Souto, C. Gómez-Amoza, J.L. Martínez-Pacheco, R. & Concheiro, A. 1999.

Interfacial Adsorption of Polymers and Surfactants: Implications for the Properties of

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Disperse Systems of Pharmaceutical Interest, Drug Development and Industrial

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Galmier, M. J. Frasey, A. M. Meski, S. Beyssac, E. Petit, J. Aiache, J. Lartigue, M. C.

2000. High-performance liquid chromatographic determination of phenylephrine and

tropicamide in human aqueous humor, Biomedical Chromatography. Issue 14, pp

202–204.



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Analytical Procedures: Methodology Q1B’, ICH Harmonised Tripartite Guideline,

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hydrochloride in pharmaceutical dosage forms with on-line solid-phase extraction

and spectrophotometric detection phenylephrine hydrochloride and orphenadrine

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Martin, M. del Castillo, A. Prados,B. P. 1993. 13-Hydrohaycena phato [1,2

b]quinoliuivs bromide as a new fluorescence indicator, Talanta. Issue 40, pp 1719–

1723.

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Olmo, B. Garcıa, A. Marın, A. Barbas. C. 2005. New approaches with two cyano

columens to the separation of acetaminophen, phenylephrine, chlorpheniramine and

related compounds, Journal of Chromatography. Issue B 817, pp 159–165

Pandey, R. K. Upadhyay, P. K. Kumar, P. 2003. Enantioselective synthesis of (R)-

phenylephrine hydrochloride. Tetrahedron Letters, vol. 44, Issue 33, pp 6245–6246.

Pandey, R. K. Upadhyay, P. K. Kumar, P. 2006. An asymmetric dihydroxylation route

towards the synthesis of (R)-clorprenaline hydrochloride. General papers, pp 10–14.

Rozet, E. Marini, R.D. Ziemons, E. Boulanger, B. Hubert, Ph. 2011. Advances in

validation risk and uncertainty assessment of bioanalytical methods. Journal of

Pharmaceutical and Biological Analysis, vol. 55, Issue 4, pp 848–858.

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188

Table 1: Accuracy data for quantification of phenylephrine hydrochloride

Theoretical

Concentration

(mg/mL)

Actual

Concentration,

Mean (n = 6)

(mg/mL)

Relative Standard Deviation (%RSD)

Percentage

Recovery (%)

9.5 9.46 0.36 99.5

54 54.1 0.02 100.1

138 136.89 0.05 99.1

Table 2: Precision data for quantification of phenylephrine hydrochloride

Theoretical

Concentration

(mg/mL)

Actual

Concentration,

Mean (n = 6)

(mg/mL)

Relative Standard Deviation (%RSD)

Percentage

Recovery (%)

9.5 9.48 0.42 99.8

54 53.86 0.06 99.7

138 137.29 0.16 99.4

189

Figure 2: Chromatogram of mobile phase alone

Figure 3: Chromatogram of phenylephrine hydrochloride only

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

190

Figure 4: Chromatogram of Product I

Figure 5: Chromatogram of Product II

0.0 2.5 5.0 7.5 10.0 12.5 min

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

mAU280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

mAU280nm,4nm (1.00)

191

Figure 6: Chromatogram of Product III

0.0 2.5 5.0 7.5 10.0 12.5 min

0

5

10

15

20

25

30

35

40

45

50

mAU280nm,4nm (1.00)

192

APPENDIX B

LIST OF EQUIPMENT

Autoclave Hirayama Manufacturing Corp, Japan

Spectrophotometer Lasec Cecil LE 2021, Wehingen, Germany

HPLC system LC2020 system, Kyoto, Japan

HPLC column Phenomenex Luna C8

PDA detector SPD M20A PDA detector, Kyoto, Japan

Incubator Labcon Incubator, Labex, Orange/grove

Edelstahl Rostfrei, Memmert, NT Laboratory

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APPENDIX C

LIST OF SOLUTIONS

Nutrient agar

Nutrient agar 16 grams

RO water to 1000 ml

0.9% Sodium chloride solution

NaCl 9.0 grams

Reverse osmosis water to 1000 ml

1 M sodium hydroxide

Sodium hydroxide 40 grams


Hydroxypropyl methylcellulose 0.3%

Hydroxy propyl methylcellulose 3 grams


Sodium carboxy methylcellulose 0.2%

Sodium carboxy methylcellulose 2 grams


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