DRUG DEGRADATION INYESTIGATION OFCORTICOSTEROID IN PRESENCE OF ANTIBIOTIC

Ph.D. Thesis

By

I]ROOJ FATIMA

DEPARTMENT OF CHEMSTRY

I]NIVERSITY OF KARACHI

PAKISTAN

SEPIEMBER,2013

*rl,

In The lvame of Allah,

The Most Beneficent

The Most Merciful

DRUG DEGRADATION IN!'ESTIGATION OF

COR"TICOSTEROTD IN PRESENCE OF ANTIBIOTIC

Ph.D. Thesis

By

UROOJ FATIMA

Submitted to Udvenity ofKarachi in tulflllment to the requircment for the degee of

+l-r-\

€

DOCTOR OF PEILOSOPIfI'

DEPARTMENT OF CHEMISTRY

UNIVERSITY OF KARACHI

PAKISTAN

SEPTEMBER,2OI3

CERTIFICATE

This is to certiry that Ms. Urooj Fatida has completed her Ph.D research work title (Drug

D€gradatiotr ltrvestigotion of Corticorteroid itr PreseNe of Atrtibiotic.in order to fulfillthe requir€ments for degee of Doctor of Philosophy. She has completed hff research workunder my supeNision. No part of this research dissertatio{ has been submifted in any oth€ritrstihrte.

Prof. Dr. Rahat Sultana

Supervisor

at

I.-2214

DEDIOATION

DEDICATED TO MY BELOVED

PARENTS, LOVINQ HUSBAND,

CARINq BR.,THEF.

AND DARLING DAIJiHTE?

CONTENTS

ACKNOWLEDGEMENT

PERSONAI- INFORMATION

LISI' OF,,\BBREVIATIONS

LIST OI TABLES

LISI'O}- FIGURXS

SUNIiVIARY

URDU KHULASA

GFNFRAI- INTRODLTCTION

il

ii

iii

viii

xviii

\xvii

r t1

t2 15i1.0

1.0

t.i

INTRODUCTION

RESUI-TS AND DISCUSSION

DEVE]-OPMENT OF UL'IRA I-AST HPLC AND TI-C

DENSITOMETRY ME'IHODS FOR DETERMINATION OI-

COR I ICOS'fEROID AND ANTIBIOTIC IN PTIARMACEUTICAI,

PRODUCTS

Validated Ultra Fast HPLC Method !-or Determination of Corticosteroid

and Antibiotic in Phannaceutical Producls

Prcdnisolone Acetate and Chlorampheoicol

l)examcthasone and Chloramphenicol

Dexamerhasone. Prednisolone Acelate and Chloramphenicol

l.i.l

2. 1.1. I

t.r.1.2

l.l.l.l

2 .l .2 Validated TLC -Densitometry Method for Determination of

Corticosteroid and Aatibiotic in Pharmaceutical Products

2.1.2.1 Prednisolone Acetate alld Chlommphenicol

2.1.2.2 Dexamethasone and Chloramphenicol

2.1.2.3 Dexamethasone, Prednisolone Acetate and Chloramphenicol

2.2 ICH RECOMMENDED STRESS DEGRADTION STUDIES AND

DEVELOPMENT OF VALIDATED STABILITY INDICATING TLC _

DENSITOMETRY METHODS FOR QUANTIFICATION OF

CORTICOSTEROID AND ANTIBIOTIC. INDIVIDUALLY AND IN

COMBINATION

2.2,1 Prednisoione Acetate ard Chloramphenicol, Individually and in Combination

2.2.2 DexamethasoneandChloramphenicoi,IndividuailyandinCombination

2.2.3 Dexamethasone, Prednisolone Acetate and Chloramphenicol, lndividually and

in Combination

3.0 EXPERIMENTAL

3.1 Calibration Standards and Sample Preparation

3.2 ICH Recommended Strcss Degradation Conditions

3.3 Ultra-Fast HPLC Instrurnentation and Parameters, Method Optimization and

Validation

3.4 TLC Densitometry Insfumentation and Parameters, Method Optimization and

Validation

4 O BIBI-TOGRAPHY

156-170

1',7 t- t78

5.0 PIJBLICATIONS 179-180

ACKNOWLEDGEMENT

I am very grateful to Almighty Allah who bestows countless boons on me at every instant, forgiven me the endurance to complete this thesis and provided me with ali oppodunitios for itssu.Lcsslul conrplction.

During the many years ofmy association with my research supeNisor, l)t. Rahal SLrltana, I

havc always beeD able 10 count on continuing suppof, help, uniquo insight and

encouragenent of hcr. She is a role rnodel for me. The intelligence, editorial skills and

research expeftise ofmy co-research supcrv;sor, Dr. Syed Ghulam MLrsharraf made this a far

better thesis and I thank thenr both for their support, wise counseling and sincere advices.

I also indebted to Dr. Iqbal Choudhary, Director, H.E.J. Research lnsfitutc of Chcnistry,International Center lor Chemical and Biological Scicnces, Dr. Sycd Azhar Ali, Chairman,

Depafnlent of Chemistry, for granting me permission to use instruments and avail each and

every lacility which is available in H.E.J. I{esearch Institute of Chemistry, InternationalCenter for Chemical ald Biological Sciences and in Department ofChemistry, University ofKarachi- I also acknowledge Dean, Faculty ofScierce, Dr. Abid Azhar for financial support.

I want to extend my warmest gratitude to Vice Chancellor, Prof. Dr. M. Afzal Haquc, pro

Vice Chancellor- l, Prof. Dr. Muzaffar Mahmood, Dean, Prof. Dr. Saroosh llashm;t l,odhiand Chaioran, Prof Dr. Ali Raza Jaferi, Department of Bio- Medical Engineering, NEI)UEI, Karachi for thcir support and co operation. I also appreciated the sincerc sugaestions ofmy collcagues at Departrnent of llio , Medical and lrood Lngineering, NED t.l B'l'. Karachi.

I \tould like to take this opportun;ty to tharlc those who earnestly helped and glrided nre ar

different stages of lny research work. It involves conce ed elforts of a nurrbcr of pcople.

They always stood beside me and a special debt is owed to all these peoplc including prolNeelofur Mastcr, Dr. Parzana Shaheen, Sadia Sultana, Ptof. Bushara Khan, Anjulr Shickh,Mumtaz Qudusi, Nooren Taj, Shawar Ahmed, Ieerven Fazil aod alt near and dear tr.iends oIrnine. I am highly thankful to all the research studcnts oI Dr. Rahat Sutrana and Dr. SycdChulatn Musharraf for tlreir l<ind supporl and help-

At home, I want to acknowledge thc efforts, support and parience of n1y beloved morhcrKhalida Ralfat Bilgrarni, who always stood beside me and madc me pers;stent and consistenttowards my studies. My father, late Mailbool-ul-Ilassan Khan Bilgrami was always a greatsource of inspiration and encouragemcnt for m€. And I have no words to acknowledge thecffol1s made by my dearest brother, Mahlnood- ul- Hassan Khan tsilgrafii who kceps a

phenomenal bord as a childhood companion witb me. Cooperation, undcrstanding arldsupport ofmy husband and my lovely dauetrter have great rote ;n compler;on ofmy studies.They all worked hard to make surc thel I couid get the quality educal;on with pcrconalizedanbieDce.

I'ERSONAL INTRODUCTION

Being the youngest child of parents and the only sister ofmy brothor, I had my brought Lrp in

very lovirg and caring hands. Passing my school and collegc levels, Itook admission in

Department of Chemislry, University of Kalachi. I was graduatcd in Dcc, 2001 afld did nyM.Sc (Organic Chemistry) io I)ec, 2002. In M.Sc Final semostcr I con'rpleted ny research

worl( based on "Microbial IransformalioD of Stcroid and lcrpcnc". This rescarch work

made Dre stood first in Poster Presentatjon Competition of M.Sc t'inal Ycar llesearch

Projects. Lator the disse ation was published as book by LAP LAMBEIU' Academic

Publishing Gmblf & Co. Believing d,at I am a bom leacher and passionate abolrt

teaching, I clloose tcachiDg as career. Besides this I was also willing to corliDuc

higher studies and for this reason I Bot enrolicd for M.Phil / Ph.D with Dr. Rahat

Sutanq my rescarch supervisor. Now it's beer ten yelrs that I am ill the llcld ofteachinB and have gaincd experience of teaching graduate, undergraduate programs

ard MCAT/ECAT in different institutes/colleges and universities. I also spent some

limc at HIIJ Research Institute of Clremistry, ICCBS and completed my rcsearch work

undcr co-supervisior, ofDr. Syed Ghulam Mushar:ral

Patience and believc ir Allah that hard work is never wastod. one should work hard.

Uneven circumstances and situations are not powerful enough to stop someone lrom

achieving the goals. It should be given that much importance that these may slow

down one's pace and may change priority order in life. 'Ihis is the persistert an.l

consistent struggle and sacrifices ofmy parents that made me able to excel.,lhey

ncver adopt any short cut that may bring short team happiness or luxuries or comfort

Lr lile.

t'My I-oard! Bestorv on thenr Your Mercy as they briig nlc up lvhen I lvas young,,

(17 : 24, Surah Bani Israel, Al-Quran)

ii

I,IST OF ABBREVIATIONS

2-amino-11,1-nitrophenyl) propane-1,3-diol

Active phannaceutical ingredients

Ad(rnosine triphosphate

British pharmacopeia

Spcrsinicol

Chloropric

Chlonnycetin

Optachlor

Cerebrospinal lluid

Ophthadex

Methadex

Spersadex

Binadex

Dexamethasone tablet

Diode aray detector

Dexoptic-C

Fluorbioptal

Deoxyribonucleic acid

Expired Spersinicol

[xpired Dexoptic - C

Electron light scattering detector

Expired prednisynth

High perlormance liquid chromatogmphy

I ligh pertbnnance thin layer chromatogmphy

Intemational conference on harmonization

Limit ofdetection

Limit of quantification

Ophthapred

Mildopred

Prcdlbrte

ANPD

API

ATP

llt,

CI

C2

Ci

C4

CSF

DI

D2

D3

D4

t)5

D.,\D

DCI

DC2

DNA

hCI

l,l)C1

l,t.st)

EPCl

IIPI,C

1lP',ll.c

ICH

I_Ot)

r.oQ

PI

P]

1,.]

ii

P4 Pred l-

P5 Ptens

PCI Prednisynth

PC2 Prednicol

Q lA (Rl) Slabilitl testing of new drug substances and products

Q2 (l{1) validation ofanalytical prccedures

QC Quality control

I{PLC Rapidresolutionliquidchromatography

l{Sll l{elativestandarddeviation

ICC Thermostatedcolumncompartment

t l.C l'hin layer chromatography

tlPI-C Ultra fast liquid chromatography

I SP United statesphannacopcia

iv

LIST OF TABLES

'lable 1 Pharmaceutical Drugs ContainingChloramphenicol

'l xhl€ 2 Pharmaceutical Drugs Contairing Prednisolone Aceiate

l ablc 3 Pharmaceutical Drugs Containing Dexamelhasone

't lrble I Pharmaceutical Drugs Contairing Chloramphenicol. Prednisolone Acetate and

De\amethasone ;n Combination

'l r b le 5 C rad ient Prograrn of U ltra Fast HPLC Nleth od for Determ ination of Prednisolone Acstate

and ChloramPheniool

'l r b le 6 Lin ear Regress ion Data of Predn iso lone Acetate and Ch loramphen icol tbr I I ltra Fast

HPLC Method

-l nbl'j 7 ,\nal-v-sis lor Repcatabilit)- and lntenncdiate Prccision ofPredrtisolone Acetalc and

Chloramphenicol

'I xble 8 Sumnary ofValidation Parameters

l'rblc t, \\.lc.n Robuslne'5 Paramellr'

'lf,bl€ l0 Rec o\ er) stud ies of Predn;so lone Acetate Pharmaceutical Samples

't able I I Recovetu- Studies olChloramphenicol Pharmaceutical Samples

Table l2 Rccovery Studies of Pharmaceutical Samples containing Prednisolone Acetate and

Chl"rarnnlrerr.iol in Cumhrnrriun

'l lrhle 1J Analvsis ofPrednisolone Acetate and Chlommphenicol Pharmaceutical Producls

.t.nblel.lCradienlProgramofUltraFastHPLCMerhodforDetenniflationofDexamethasoneand

Chloramphenicol

'table 15 Linear Regression Data of Dexamethasone and Chloramphenicol for Ultra Fast HPLC

Method

'I rble 16 AnalJ'sis fbr repeatability and Intermediate Precision ofDexamethasone and

ahloramphenicol

Table 17 Summary of Validation Parameters

'Itlhk 18 Reco!cry StLIdies ofDexamethasone Pharmaceutical Samples

lrhle l9 {eco\ery Studics oi Pharmaceuticil Samples containing Dexamerha\onc and

lhloramphcnicol in Combination

-Irbk 20 ,Analysis olDexamethasone and Chlo.amphenicol Pharmaceutical Products

'l abl€ 2l Cradient Program of Ultra Fast HPLC Method for Determination of Dexamethasone,Prednisolone Acetate and ChlorampheDicol

lnble 22 Lirear Regression Data of Dexamethasone, Prednisolone Acetate and Chloramphenicoltbr Ultra Fast HPLC Method

'l nblc 2J Arallsis lor Repeatability and lntennediale Precision of Dexametnasone, Prednisolone

acetate and Chloramphenicol

Txble 2,1 Summary ofValidation Parameters

l rhl€ 25 Analysis of Dexamethasone, Prednisolone Acetate and Chloramphenicol Pharmaceutical

Products

'l'able 26 Opti,rizarion Data ofPrednisolone Acetate and Chloramphenicoi for TLC DensitometryMerhod

-l able 27 l.inear Regression Data ofPrednisolone Acelate and Chloramphenicol for TLCDensitometry Method

'trble28 Summary ofValidation Parameters ofPrednisolone Acetate and Chloramphenicol forILC Densitomeiry Method

Table 29 Summary of Robustness Parameters

Trble 30 Reco\,ery Studies

'l nhlc J I '\llal)sis (rl Pharmaccu.ical Producls

'I rbk 32 Optim;zation Data ofDexamethasone and Chloramphenicol fbr TLC Densitometry

Method

'l rble 33 Linear Regression Data ofDexamethasone and Chloramphenicol for TLC DensitometryMethod

'Irbl€3{ Surnnrary of Validation Parameters of Dexamethasone and Chloramphenicol for l-LCDensitometry Method

-l able l5 Surrnrar) ol Robustress Paranreters

I nbl€ 36 Recovery Studies

l'rhle.l7 Analvsis of Phannace utical Products

I rblr 3lt I-incrr Rcgression l)ata ol Dexamethasonc. Prednisolone Acetate 3nd Chioramphericolior l l.( Dcnsltometr\ Uethod

'I nhl€ 39 S mrnary ofValidation Paranreters of Dexameih asone. Prednisolone Acetate and

Chloramphenicol fbr TLC D€nsitometry- Method

vi

Tnble {0 Recovery Studies

'lable {1 Anal}sis of Pharmaoeutical Products

'lable:12 Linear Regression Data and Validatior Parameters olPrednisolone Acetate and

Chlora phetricol ibr ILC Densitomc() Melhod

I rblc lJ Summary ofsrress DegradatioD Studies of Prednisolone Acetate and PrednisoloDe

Acetate and Chloramphenicol ;n Combination

'table,l.t Summary ofStress Degradation Studies olChloramphenicol and Prednisolone Acetate

and Chloramphenicol in Combination

'l able ,15 Linear Regression Data and Validation Parameters ofDexamethasone and

Chloramphenicol for TLC Densitometry Method

Table {6 Sumnlary of Stress Degradation Studies of Dexamethasone and Dexamethasone and

Clrlordmnherri(^l irr'ombinJti^n

'I rhle .t7 Summary of stress Degradation Studies ofChloramphenicol and Dexamethasone and

( 'lrlordmplrenr'ol in ( ombinaliorr

't able 48 Linear Regression Daia and Validation Parameters of Dexamethasone, Prednisolone

Acetate ard Chloramphenicol fbr TLC Densitometry Method

l rblc {9 Summary ol Stress Degradation Stt,dies of Dexamethasone and Dexamethasone_

Prednisolone Acetate and Chloramphenicol in Combination

Table 50 Summary of Stress Degradation Studies ofPredflisolone Acetate and Dexamethason€'

Predllisololre Acetate and Chloramphenicol in Combination

'I able 5l Summary ofstress Degraclation Studies ofChloramphenicol and Dexamerh'sone

Prednisolone Acetate and Chloramphenicol in Combination

I,IST OF FIGUR.ES

Figure I Chemical Structures of(n) Dexamethasone (b) Prednisolore Acetate and

(c) Chlora,npheDicol

!'igure 2 Calibration Curveof Standard Prednisolone Aceiate for Ultra Fast HPLC Method

l'ig re 3 Calibration CuNeof Standard Chloramphenicolfor Ultra Fast HPLC Melhod

fitaur': -l Chromatogram of Standard Chloramphenicol and Prednisolone Acetate in Combination

Simultaneously Detected at ].rnax of 243 nm and 2?8 nm Resp€ctively lhrough DAD

L)etcctor

t'igure 5 Chrornatografirs of Pharmaceutical Products Containing Prednisolone Acetate and

Chlorarnphenicol in Combinatior alongwith Standards

l-igure 6 Chromatogram ofPrednisolone Acetale Pharmaceutical Products alongwith Standards

t'igure 7 Chromatogram of Ch Ioramphen ico I Pharmaceutical Products alongwith Standards

t'i,.Iur€ 8 Chromatogram of Standard Chloramphenicol and Dexamethasone in Combinalion

Simultaneousl) Detected through DAD Detecror

!'igure 9 Chromalograms of Pharmaceutical Products Containing Dexamethasone and

Chloramphenicol ilr Combination alorgwith Standards

t.igur0l0ChromatogranofstarrdardDexamcthasone.PrednisololleAcetateandChloramphcoicolin Cornbinalion

!igurell Chromatogram of staDdard Prednisolone Acerate (a), Chlorampbenicol (b) and

Prednisolone Acetate and Chloramphenicol (c) in combinatioo

t'igure 12 TLC Pla:e showing the spots of standard Prednisolone Acetate. Chloramphenicol aod

their individual and combined Pharmaceulical products

figurel3ChlomatograDofstaldardPrednisoloneAcetate,Chloramphenicolandthe;rcombinedand iDdividual pharmaceuiical products

Figure l{ ILC plate showirrg spots of standard Dexamethasone (a). Chloramphenicol (b) artd

Dexamethasone nnd Chloramphenicol (c) in combination alongw'lh their pharmaceutical

prtxlLrcis

l'ignrc 15 TI-C plate showirlg spots of standard chloramphenicol. prednisolone acetate'

chlorarnphenicol and prednisolone acetate in combination (a)1N HC l. 5N HCl 0 lN

NaOtl. lN NaOH. 5N NaOH (b) neutral hydrohsis, wet heating- dry heating photo

desradation and oxidation at room temperature

l'igure 16 Chr(inrarograrn ol prednisolo|e acelarc al ),ma\ 24:jrrD (t). chloramphenicol at inia\218 nm (2) and prednisoloneaceute and chloranphenicol at).278 nm in combiration (3)subjected to acidic hydrolysis ( lN HCI)

!igurc 17 Chromatogram olprednisolooe acelate at).max 243nm (j). chloramphenicol at J"max 278nm (2) and prednisoloneacetate and chlorafiphenicol at I 278 nm in combination (l)subjected to alkalinehydrclysis (0.1N NaOH)

Chxrmatogram oI predrrisolone acelrle at ima\ 2:llnm ( l), chlo.amphenicol atima\ ll3Drn (2) and prednisoloneacetate and chloramphenicol at ).278 nm in combination (3)

snbjected to neurral hydrolysis

Chronratogram of prednisolone acetate at imax 2a3nm (1). chloramphenicoi at ima\278 nIn (:) xnd prednisoloneacetate and chloramphenicol at i278 um in combiralion (3)

sLrbicctcd lo wer heat

Chromarogram of prednisolone acetate at }"ma\ 2.13nm 1l), chloramphenicol at imax173 lln (2) and prednisoloneacetate aDd chlommphenicol a! i 278 nm in conrbination(])sub jected to photodegradation

Chrcmatosram of prednisolone acetate a! imax 2a3nm ( 1), chloramphenicol at )"max

118 rrnr (3) and prednisoknrercetate lId chlorarnphenicol at i]78 nn in combinarim (l)subjected to oxidation atroom temperallrre

Chromatogram of (a) Expired Spersinicol and (b) Expired Prednisynth at r" 278 r]m

ILC Plate showing spots ofstandard chloramphenicol. dexamethasone, chloramphenicol

aDd dexamethasone in combination- in ]N HC l, 5N HCl. 0.1N NaOH, lN Naotl.5NNaoH. neLrtral hydrolysis. oxidation and photodegrdadation

Tt-C Plare showing spots of standard dexamethasone, chloramphenicol, prednisolone

acetate, acidic hydrolysis, alkaine hydrolysis, neutral hydroiysis, wet heat dcgradation

and photodegradation

I igure l8

fiqure l9

F igure20

F igu re 2l

!'igure 2{

l-ianre l2

l'igu re 23

ix

SUMMARY

This I'h.D- dissertation.is si.ucrured into two parlst firsr part is based on developnlcnr ofvalidated Ultra Fast HI,LC and TLC Densirorretry merhods for determination ofconicosteroid aDd antibiotic h pharmaceutical products (prednisolone accrate,

dexamethasone and chloramphenicol). Second part states the ICH recommendcd stress

degradation studies and development of validated TLC Densjto,netry mcthods for

quantification ofcorticosteroid and antibiotic, individually and in colrbination. Appjied slress

conditions involvcd acidic hydrolysis (l and 5 N HCI), alkaline hydrolysis (0.1, I and 5 N

NaOtl), neulral hydrolysis, lvet heat and.dry heat degradation, oxidation rcaction (J5% v/v

Il2or, photodogradatioI and buffer solutiorl.

Pharmaceutical products subjected to analysis includcs Chloromycetin (capsule), Chloroptic

and Spcrsinicol eye drops containing Chloramphenicol; Pred+, Prens, predforte. Opbthaprcd

and Mildopred (ophthalmic suspensions) containing Predoisolone Acctarc and Ophth dex

and Bnladcx (drops) containing Dexamethasone and Dexamethasore Tablets. phamaceurical

products analyzed containing corticosteroid and antibiotic in combination were Prednisynth

and PredDicol (Prednisolone Acetate and Chloramphenicol); Dexoptic-C and Fluorobioptat

(Dexamethasone ard Chloramphenicol).

Most 01'th€ conventional HPLC melhods have been shifted to the ultra-fast IIpl-C ncthods

Ior thc analysis of phannaceutical products due to its high throughput and rcduced cost.

Morcovcr Tl-C - Densitonlclry has also got tnajor attention. in tbc ficld duc 10 ils low oost

and the fact ofanalyzing many samples in a singlc run. J-aking this aspcct in consideratiotl,

the thesis involves:

A (i) Development and Validation of Ultra Fast HPLC McthodsPrcdtrisolone Acct,tc and Chloramphcnicol in Combinxtion

The ultla-fast HPLC melhod was devclopod on RP-CI8 column having particle size oI l.B

|lm in run tine oI3.5 minutes. Chloramphenicol ard prednisolone acetate wcrc cluted at 1ir of0.648 r-0.0133 and 0.888-r 0.0097, respectively using linear gradient mobilc phase

composed of acetonilrile and water- Analytes werc detected by photodiode array dctcction at

multi wa\relengths of 243 (),,,"- ol Prednisolone Acetate) and 278 nrr (],max ol

Chlolamphenicol). The response uas linear over the concentration range of 50-1000 pg/ml,

r,rith correlation coeflicient of 0.9999 and 0.9996 ibr Prcdnisolone Acetate rurd

Chloramphenicol- respectively. Following the recommended ICH guidelines. the developed

mcthod was validated for linearity- accuracy. specificity, robustness. LOD and LOQ. The

accurac) was above 94 % for inter-day emd 9i% ibr intra day precision. The %RSD is found

10 bc lcss than I % in all inter clay and intraday results. For Prednisolone Acetate. LOD is

lbund to be 6.689 ng/gl and LOQ was 20.2'7 nglp.L while for Chloramphenicol. LOD is

[bund to be J6.893ng/prl and LOQ was 111.79n9/pl. Method was robust and

specilic.Developed method was found rcliabie tbr quality contol laboratory analysis and was

successfr l-v applied lor the detection of wide variety of pharmaceutical products (eleven)

includin-t u1_e drops. capsule and ointment in individual and combined pharmaceutical

products containing PrednisoloDe Acetate and Chlo6mphcoicol.

l)examethasone and Chloramphenicol in Combination

I-IPLC method was developed on Zorbex Eclipse XDB RP-C18 column (50 x 4.6 mm I.D. 'l.ll pm) in3-0 minutes run time. linear gradient mobile phase of acetonitdle and water.

in\olving multiwavelength detection at 238.5 and 278nm. Tu'o peaks at npf 2.550 10.0060

and &2.802 ;0.0065 were identified in chromatogram as Chloramphenicol and

l)e\amethasone in comparison to standard chromalogram The developed method was

validated as per ICH guidelines for inter day and intraday precision, accuacy, linearity,

speciticity. roblLstness. I-OQ and I-OD. Linear response was achieved within the

concentration range of 50-1000 pg/ml. showed corelation coeflicient of0.9998 and 0.9997

tbr Dexamethasone nnd Chloramphenicol respectively. The accuracy was above 98 7o lbr

inter day iuld 99 9'o lor intra day precision. The %RSD is lound to be iess than 2 9'n in all inter

rlay and intraday results. For Dexamethasone. LOD and LOQ vvere found to be14.397 ng/pl

and 4i.632 ng./pl while for Chloramphenicol, LOD and LOQ were for.rnd to be 37.013 ng/pl

and lll.161 og,1pl. Method was robust and specific.Method was successfully applied on ten

pharmaceutical preparations of Dexamethasone and Chloramphenicoi including four eye

drops and a tablei lormulation of Dexamethasone: two eye drops and capsule of

(ihloramphenicol; and eye drops containing Dexamethasone alld Chloramphenicol in

comhination and was tbund reiiable for routine laboratory analysis.

Dcr(ameth,rsone, Prednisolone Acetate :rnd Chloramphenicol in Combination

-lhc proposed Ultra rast HPLC mcthod was developed on Zorbex Eclipse XDB l{P-Cll3

collrmn (50 x 4.6 mm l.D. , 1.8 pm) ir total run timc of 4-0 minutcs rrsing lincar gradicnl

mobile phasc composed of acetoritrile and water involving mLllti photodiodc array dctectiol

at 25,1 nm. Thlce pcaks at Fr of I-496 t 0-0042 was recognized as Chlorampltcnicol, pcak at

-Rr of2.186 t 0.0016 was rccognizcd as dcxamethasone whilc pcak at -Rr of2.685 ! 0.0024

rvas rccognized as Prednisolone Acetate iir comparison to standard chronratogram. Following

the rccommended lCll guidelines for validation of dcveloped rnethod. linearitl'. accuracy;

intlrr day and intradey precision, specificity, robustness, LOD and I-OQ were validated. The

response was linear over the concentratior rangc of 50-1000 psrnl, and correlation

coelficient of 0.9999, 0.9999 and 0.9997 for Dexamethasonc, Predtr;solone Acctate and

Chloramphenicol respectively were obtained. Following the rccommcndcd ICH guidclircs,

the developed netho-d was validated for Iincarily, accuracy, specificity, robustncss, LOD and

I-OQ.'l'he accuracy was above 97 70 for both intra day and inlcr day prccision.'l'hc %RSD

is found to bc icss than 2 % in all inter day and inlraday resLrlts. For Dexa,nethasone. I-OD

and LOQ in ng/pL w€re lbund to be 14-684 and 44.195 while tor Prednisolone Acelato thcse

were 6.689 ard 20.271 respcctivcly. For Chloramphenicol, LOD and LOQ were 17.763 and

I11.,+32. Mcthod was found reliable ior QC laboratory analysis and successfully applied on

seventeen pharmaceutical products including Dexamethasone eye drops and tablct;

Prednisolonc acetate eye drops; Chloramphonicol oyc drops and capsLrle; and eye drops

containirg Dexalnethasone and Chloramplrenicol, and Prednisolone Acetato and

Chloranphenicol in combination.

A (ii) Dcvclopmcnt and Validation of TLC-Densitomctry Mclhods

Prcdnisolonc Acetate and Chloramphenicol in Combinatior

Separation lvas done on ILC glass plates, pre-coated with silica gcl 601r,254 using

chloroform: nrcthanol (14:1 v/v). Spots at,4r 0.21 + 0.02 and 0.41 r 0.03 were recog,rized as

Chloramphenicol and Prednisolone Acetate, respectively. Quanli(ative anatysis was done

through donsitometr;c measurements at mult;wavelength (243 nm, L.a, of prednisolonc

Acetatc ard 278 nm, )",,,"- oI Chloramphcnicol), simultaneolrsly. Thc dcvctoped mcrhod was

optimized and validated as pcr ICH guidelincs. Slandard calibratior curve lor bolh

I'rednisolonc Acetate and Chloramphenicol io rhc concentration rangc of 200-6000 ng/spot

wrs found lirear with .'?1S.O. O.lleO 10.035 and 0.9920 10.025, r€specrivety. Intra-day

prcoision lbr Prednisolone Acetate and Chloramphenicol *ere found to be 1 :12 and i 09

whileinter-day precision was found to be i.90 and 1.68, rcspectively in terms of % R S D'

lror Prednisolone Acetate LOD and LOQ were found to be 0 o477nglspotand

(). I 4,16ng/spot$ hile lbr ChLoramphenicol these were found to be and 00300 ng/spot and

0.0912 ng/spot. respectively. Method was robust- specific and accurate The developed TLC

method can be applied tbr routine analysis of Prednisolone Acetate and Chioramphenicol in

rhcir individual and combined pharmaceutical formulations

l)examethasone and Chlorampheni€ol in Combination

,\ simplc and accurate HPTLC densitometric method was developed for detemination of

I)cxanethasone and Chloramphenicol in pharrnaceuiical preparations Samples were spotted

on LIP I LC silica gel plates 60 F 254 and were developed in cblorcform: acetone (8:2) A1ier

dcvelopment. Dexamelhasole was lbuld at Rr0.404] 0 027 and Chloramphenicolwas found

at 0.51,110.026.For quantitative analysis densitomeldc measuremerlts of these spots were

Jone at multirvavelength of 238.5 nm and 278nm Following ICH Guidelines Q1AR2' the

.lc\.elopcd mcthod r-r'as validated. Merhod u'as ibund linear tbr both Dexamethasooe utd

Chloramphenicol in the concentration range of 200-6000 ng/spot wirh 12i S'D.

()t)909-0.002and0,994610,0021respectivel-v.rorlnua-dayandintel.dayprecision.To

R.S.D. observed lbr Dexamethasone uas 4-62 and 3 16 respectiveiy whiie for

('hloramphenicol. 2.87 and3.'16' respectivel-v' For Dexamethasone and Chloramphenicol'

l.OlJs r,,erc lbund to be 0.0049nglspot and 0'0051ng/spot, respecdvell while LOQS were

tiruid to be 0.0149ng/'spotand 0.0153ng/spot' respeclivel,v Method was accurate rcbust and

acculare and el'tectiYely separates both the oomponents in pharmaceutical productscontaining

these active components.

I)cxamethasone, Prednisolone Acetate and Chloramphenicol in Combination

:\ sirnple and accurate HPTLC densitomet c melhod was developed for determination of

I)cxamethasone. Prednisolone Acetate and Chioramphenicol in phalmaceutical prepalations

Szr,nples u'ere spotted on HPTLC silica gel plates 60 F 254 and were developed in

chlorolorm: mcthanol (14:1 2nd run). Affer development, Dexamethasone was foutd at Rr

value o1 0.l8t o.o2.Chloramphenicolat 0'21 i 0'01 and Prednisolone Acetate at 018 +

0 02.For quantitative zuralysis densitometric measuements of these spots were donc at

multi*avelengrh of 238.5 nm. 243 nm and 2T8nm Standard calibration curve for

I)cxamclhasonc. Prednisolone Acetate and Chloramphenicol in the concentuation range of

xiii

was lound linear with 7"'z1S.D.0.9966 10.015, 09920.!0.026 and 0.9981 + 0.024

rospcclively. For Intra day and inter-day precision, % R.S.D. observcd lbr Dexancthasonc

was 3.87 and 2.91, fbr Prednisolone Acetato 2.38 and 3.04 while lor Chlorarnphenicol, 2.19

and 2.08, rcspectively. For Dexamethasone, Prednisolone Acetate and Chloramphenicol,

LODs were found to be 0.088 ng/spot, 0.035 ng/spot and 0.029 ng/spot, respcctively while

LOQs were found to be 0.269 ng/spot, 0.107 nglspot and 0.088 ng/spot, respcclivcly. 'lhc

developed TLC rnelhod was foUnd robust, specific and accumte and can bc applicd for

routinc analysis of Dexamethasonc, Prednisolone Acctate aod Chloranrphenicol in

pharlnaccutical forrrulations.

ll ICII Rccommcndcd Strcss Degradation Studics and l)cvclopmcnt of

Validated Stabili[, Indicaling TLC - Dcnsitomctry Mcthods

l'rcdnisolonc Acc(ate and Chloramphenicol in Combination

A rapid and reproducible stability indicating TI-C method was developcd for the

determination of Prednisolone ,{cetate and Chloramphcnicol in presence oI their degraded

pfoducts. llniform degradation conclitions were maintained by reflLrxing sixtccn rcaction

f ixtures lor tNo hours at 80"C using parallel synthesizer including acidic, alkalinc and

ncutral hydrolysis, oxidation and wet heatirg degradation. Oxidalion al room tempcrature,

photochenical and dry heating degradation sbdics were also carricd ollt. Scparation \.vas

donc on 'lLC glass plates. pre-coatcd w,ith silica gcl 60F 254 using chlorofol,lni methanol

(14:l v/v). Spots at 1?f 0.21 + 0.02 and -Rr 0.41 + 0 03 we.e recognized as Chlorarrphcnicol

and Pr€dnisolone Acetaie, respcctively. Quantitative analysis was done through .lensilomctric

measurements at mulii wavetength (243 nm, 7,,"- ofPrednisolone Acetatc and 278 nm, ),marol

Chloramphcnicol), simultaneously. The developed method was optimized and validated as

per tCIl guidetires. Method was found linear over the concentratiorl range of 200 6000

ng/spot wilh thc corrclation coefflcienl (r2 + S. D.) of 0.9976 + 0.035 and 0.9920 r. 0.025 for

Prcdnisolone Acclatc and ChloraNphenicol, rcspcctively. Acidic, alkalirc alld nelltral

hydrolyscs coiditions, wet heat and photochemical condilions showed greater degradation

offocts to Prcdnisolone Acetate alonc. Chlorarnphenicol, alonc showed grcater susceptibility

to degradation to alkalino, wet heating and photochemical stress conditions. ln combination,

Prednisolone Acctatc was degraded morc under acidic hydrolysis aDd oxidation rcaction

whilc Chloramphenicol was degraded high undel all stress conditions in conbination except

photo degradation condition. Thc developcd'fLC mclhod can bc applied lor routinc analysis

anallsis of Preclnisolone Acetate and Chlorampheoicol in presence of their degBded products

in their individual and combined phamaceutical fotmulations.

l)cxamethasono nrrd Chloramphenicol in Combination

r\ rapid ancl reptoducible stability indicating TLC method was developed for the

determination ofDexamthasone and Chloramphenicol in presence oftheir degraded Products'

tlnifbm degradation conditions were maintained by refluxing sixteen reaction mixtwes for

1\\o hours at 80oC using parallel synthesizer including acidic, aikaline and neuhai hydrolysis,

o\idalion and wet heating degradation. Oxidation at room temperature. photochemical and

Jr) hcrling degradation sludies were also cartied out samples were spotted on HP I LC silica

gel plates 60 l, 254 and were deveLoped in ohloroform: methanol (14:1 2nd run) After

developmenr. Dexamethasone and Chloramphenicol were found at Rfvalue of0 1810 02 and

0 l1 I 0.015. respecrively.Quantitative analysis was done through densitometric

Drcasuremenis .rt multiwavelergth (218.5 nm. i,n* of Prednisolone Acetate and 278 nm.

).,,,,of Chloramphenicol). simultaneously. For both Dexamethasone and Chloramphenicol'

rncLhod $,as iound Linear in the concenlration range of200-6000 ng/spot with r21 S D 0 9966

r0.0i5 and 0.998110.024. respectively.Acidic. alkaline and neutral hydrolyses conditions'

\\eI hcatand photochemical conditions showed greater degradation el'fects loDe'xameihasone_

alone. Chloramphenicol. aione shou'ed greater susceptibility to degradation to alkaline' wet

huating and photochemical stress conditiolls. In oombination. Dexamethasone was degnded

urorc in comparison to stress applied individually while chloramphenicol showed higher

degradalior under a1i stress conditions except dry heating and photo degradation Developed

fl.( method can be applied for anal)sis of Dexamethasone :!nd Chloramphenicol in prescnce

oftheir clegraded products in the individual and combined pharmaceutical formulations'

I)rramethasonc. Prednisolone Acetate and Chloramphenicol in Combination

A rapid and reproducibie stability indicating TLC method was developed for the

determination of Dexamethasone. Paednisolone Acetate and Chloramphenicol in presence of

rhcir degraded products. Unilbrm degmdation conditions were maintained by refluxing

rLjitcrion rrixrures fbr lwo hours at 80.c using parallel synthesizer including acidic. alkaline

and Dcutral hydrol"vsis. oxidation and \\'et heating degradation' Oxidation at room

tcmpcrxtLrrc. photochemical and drl- heating degradation studies were also caried out'

Suitable scparation *ith best resolution was achieved with chlorofom I methanol (14 | 1

v'v2"' r-un) rvhich showed sharp bands with Rt valueof Chloramphenicol at 021 I 0'02'

l)cramethasor,e al 0- 181 0-02 and of Prednisoione Acetate at 0 41 : 0 01'Standard calibration

clnve otl)examethasone.Prednisolone Acetate aod Chloramphenicol was found linear in ihe

conccntratjon range o1 200-6000 ng/spot with rr1 S.D 0 9966 10 029, 0 998910 033and

0.9920 l-0.02,1. respectively. Quantitative analysis was done through densitometric

nleasurements at multiwavelength (238.5. 243 nm and 278 nm), simultaneously The

clereloped method lvas optimized and validated as per iCH guidelines Comparative study

showed grearer tiegradation of dexamethasone under all conditions except acidic hydrolysis'

neutral hydroiysis and dry heating condittons. Ior Prednisolone acetate higher degradation

was ohsenecl under all conditions except neulral hydrolysis. wet and dry heating and photo

.lcgradalion conditions. Chloramphenicol showed higher degradation under all conditions

.\cepl .lry heating and photo degradation. The developed TLC method can be applied for

routine anal)sis of Dexamethasone. Prednisolone Acelate and Chlorarnphenicol in presence

of th.ir degraded producrs in their individual antl combined phamtaceutical formulations'

rr H l-1

: H

(b)

NH ,CHCI2

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(c)

Figure

Chcr.nical tructures of (a

xvii

Chloramphenicol

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GDNERAL INTRODUCTION

Centuries earlicr, humans had learned to use crude prcparations empirically for the iopicaltreatulent of infections. Following the rapid progress of medicine and phanraceuticalsciences, mankind overcame illness and controlled their own destiny through science andtechnology and thus quality oflife arrd well being have been greatly improved.

While modern science has Ied to the development of highly researched and standardjzed

medications, leading to rhe isolation of hundreds ro rhousands of natural product srructlrres,only a srrall number of srructurar tvpes have proven erficacioLrs a,d safe cnoush to ht:

approved lor human cliDical use to t.eat bacterial diseases.

Drugs are no loDgerjust tbe drugs but thesc are prodLrcls dcsigned with a specific uso in lnindand contair many orher conponents besides 1he active componcnt, having quite complicalodchernical structnres and are by definition biologically active compollnds. lt should not.therefore come as surprise that these reactive molecules Llndergo chemical reactions whichresult in tlreir decomposilion aDd deteriomtior and ihat thc ptocesses bcgin as soon as thedrug is syrthesized or the medicjne is fbrmulated.

As the intensity and duration ofdrug action is proportional to the collcentmtior ofthc drug, adrug must be plesent in appropriate concenkation at its site of action to produce itscharacteristic eflects. Therefore any factor that effectivcly alters the drug concenh.ation at theactive site lvill result in a changed pharmacological response to drug.

ln cotnparison fo solid dosage forms, prolonged storagc of organic conrpounds in solutionform can lead to significant dcgradation. In add;tiol1, a variety of cnvironmental lactors are

also responsible lor effccting the compound stability that results jn decl;ne ofrhe anatyricalquality of the drug active components includes hurnidity, tempcrature, oontainer nrateriar.

storage format, storage time, hydrolysis, oxidarion, sun lighr and pH.

Pharnaceutical preparalions containirg combination of corticosteroid and anribioric are*,idely practiced for lreatment of superficiat eye inFections. In various cases, rhesecornbinations (Chloramphenicot and Dexamethasone, Chtoramphenicotand Dexameihasone

Sodiufi Phosphale. Chloramphenicol and Prednisolone Acetate. Chlorarnphenicol and

I l-,'dK)col tisone Acetale. Chlorampl, enicol and Betamethasone etc.) are proved more etlective

than tl'ie antibiotic or corticosteroid alone. Man)-, of rhese combinations are not official with

1.,'SP. BP or European Pharmacopeia. Unfavorable climatic conditions leading to degradation

of thesc drugs may cause cenain hamfui effects on eyes. Shelf life deterioration of these

rlrugs rre mainlv due to decomposition reactions brought about by heat. light, ahnosphere.

oridation and hydrolysis. Stability testing is done to provide evidence on how the quality ofa

(lrus substancc or da.rg paoduct varies wirh timc under the influence of variety ot'

cnvironmental lbctols such as temperature. humidit) and light.

Slress testing of the dmg substance can help identily the likely degradation products.

l:xamining degradation products under stress conditions is usei'ul in establishing degradation

path$a,!-s and developing and validatirg suitabie procedures lbr.u1alysis. ICH recommended

strcss degradalion conditions requires ihat acidic. alkaline and neutral hydrolysis, wet and dry

hcat degmdation. oxidation and photodegradation reactions be performed. In order to suppon

stability iesting prolocols: generally practiced tecbniques are Dissolution. Gel

lrlcctrophoresis, GC. HPTLC- DSC, HPLC, and X- Ray Powder Diffraction. Among all

rhcsc analytical techniques. chomaiogiaphy pafiicularly HPLC and HPTLC have become the

llrost inrportant and tront line techniques for the characteaization ofdnrg.

lhc parent drug stabilit."- test guidelines (QlA) issued by ICH requires that anal)'tical test

proccdu.es should be i'ully validated and the assa) should be slabiliry indicating. Validation

faramctcrs fbcused should invohe linearily. accuracy. precision (repeatability and

rc produsibiii!y )- specit-lcit!. robustness. limjt ofdelcction and limit ofquantificadon.

Valid anal)-tical method with adequate sensitivit). specificitv and accuEcy helps to analyze

the dmg components in pharmaceutical preparations. Most of the pharmaceutical analysis

made application ofraditional HPLC system- Literature suNey revealed that a large number

of RP-IIPI-C tcchniques are repofted for the analysis of corticosteroid and antibiotics either

scparatcly or in combination. in different types ol samples [7-26]. To meet the demand lor

rapicl auralysis of pharmaceutical products. ultra-lilst HPLC method has received great

x[cnrioD due Lo ils phcnomenal perfomance and high $]oughput. Now a cultue of

rlrnslilring lll'l.C tcchnique to lLLtra-iast IIPI-C has been adopted and is practicing

rvorlclu.idc. It is :r relativeiy neu tcchnique oi chromatography that in comparison with t{Pl-C

xxviii

opemtes at high pressures up to 15000 psi. reqlires less volume of sample and captues

dctecbr signals at high data rates lbr fast eluting peaks. Due to irs high sensitiviry-. reduced

run time. less carryover and injection volume, Iess solvent consumption and column re-

equilibration time. it results in low operational cost and t'aster anaiysis with increased

resolution and sensitivii.v.

Many antibiotics and corticosteroids are separately analyzed through ultra-fast IIPLC

methods. but literatue revealed that no attempt is made so far for the aaalysis of

corticostercid ?rnd antibiotic in pharmaceutical preparations though ultm-l'ast HPLC in

conbinalion in clinicalll used products. Fe\l HPTLC methods are reported lbr the

dctcrmination of Chloramphenicoi in combination with Benzocaine and ANPD. and lor

Jitferent corticosteroids including Hydrocortisone. Hydrocortisone Acetate, Prcdnisolone,

Ilctaructhasore l7-Valerate. Prednisolone Sodium Phosphate. Dexamethasone Sodium

l'hospilcte and lletamelhasone Sodium Phosphate. No reports ibr the simultaneous

dctermination ol Dexamethasone and Chloramphenicol. Dexamethasone Sodium Phosphate

and Chloramphenicol and Prednisolone Acctatc and Chloramphenicol in prcsencc ol. thcir

dcgraded products tlrough TLC densitometry is found.Validated stability indicating ultra

t'ast I{PLC or TLC - densirome!ry methods for determination of these drugs in combinalion

arc also not repofted so l_ar.

Taking tCH rccommendalions in consideration. individual steroids and antibiotics and

combiialions of cofticosteroid and amibiotic were subjected to the vz! ety of suggested stress

test conditions to estabiish inherent stability of the drug and to develop validated stability

indicatinsullra IasIIIPI-(l and ILC densitometry methods for determiradons of thesc

rorticosteroid and antibiotic in presence ofdegraded products.

1.0 INTRODUCTION

1.I DRUGS

Any chemical agcDl \\rhich is capable ofproducing pharnlacological activity, aflircting livirlg

protoplasm, preventing or diagnosirg diseases is called drug (drogue= dry herb).

A drug produot is the finished dosage ibrm

associaled with non-drug components, conatins

inactive ingredients that makoup the vehicle,

substancestrl.

(e.g. capsule, tablet, ointment) gcnerally

the API (active pharmaceutical ingrcdienls),

formulation and other excipienl or inert

1.1.1 flistorical Background of Drugs

A long history is associated with the drug discovery and development and dates back to the

early days ofhuman civiiizations. In acient limcs, drogs were practiced as physical remedies

and involved associatio! with religious and spiritual healing. Many of the extensively used

drugs today have been known for hundreds or thousands ofyears. Hisrory reveals that rhesc

drugs were discovered accidently through combinations of trial and error experimentation

and by observing human and animal reactions, generaiod after ingestjng thcsc drug

sub-Lan.cslzr Fe$ of rhc<c in.lude eplredrioc, reserpine, caffeine, opium c1c. Ephedrine was

used in China for over 5000 yearsirl.

Art ofDivination and Library of King Assurbanipar

The ad of divination is flrst loown to be used in Bby Lonion Assyrian medicine in

association with astrology to dcteunine thc influence of Stellors constellations on hunlan

w€lfare and mcdical ethics. The library of King Assurbanipal showed 250 vcgetable and l20

mineral drugs. identified in the clav tabletstil.

llippocratcs, Ii'athcr of Mcdicinc

A major and anonymous writing, Coprus l-lippocraticum considers Ilippocrates (:-460 - 377

BC) as lhc lathcr oi meilicjne. An antlpoison, nndrorna(hus rvas imp|overj by a Roman

Nero's I'hysician that incrcascd tho nuftb€r of toxic ingredieDts fron 54 to 70, dcscribcd in

Phalmacopoeias for'ceitturies under the name ofTheriac tal.

Egyptian Medicinc

Records olcarly tsgyptian Medical knowledge arc provided by Ancient Papyrus in black and

white.877 prcscriptions lor intcrnal medic;nes, eye, sl<in problcms and gynecoloey ^re

lound

frorn around 3000 BC in Thc Dbcrs Popyrus l'1l. I

Chincsc Medicinc

Tradaitional Chinese Medicine is believed

emperor Sheng Nong in 3500 BC t'].

Indian Medicinc

be originated in thc tinlcs of thc lcgcndary

In ancicnl times, Brahmin Sages were used to of practicing Ayeur Vcdic, thc Iodian folk

medicine, tEced back to 3000-5000 years. J'he keatmenls were set out in sacrcd writings

callcrl Vedast2l.

Grcc|(s Mcdicinc

Dispelling the ration, "diseases are due to super natural causes" is considered to be thc

greatcsl contribution of Greeks to the ficld of medicine so far. Ir Grcck Dylhology, Cod

Asclcpius was considered to be the first physician, involved in healing. Asclcpius achievcd

his reputatior by rrastering ihc experlese of surgcry and the healnrg miracles of medicinal

3

Roilans and Arabiaas ftiedicine

The two most promift.nt fi gures it tnedical history of Rofre whose co[tribution rcmain the

uncontested "standard' lot bctany and medicine are Discorider and Galenlal. Arabian major

contribution is the knor le.lge oi medical preparations and distillation method l2l.

ilIuslims Meclicine

During Middle Ages (.100 - I:00 AD), i,iuslim Empires of South West and Cenhal Afiica

made significant contributi()n! to medicin(,.Rhazer, a Persian bom physician of late 800s and

early 900s wrote the firct accur atc descriptions of measles and small pox. Avicena, an Arab

physician of late 900s an,1 eari 7 1000, wrote a vast medical encyclopedia called anon of

medical Itr.

Affer World War l, the nrodcm p,lamaceutical indusa.y came into existance and drug

discovery and development, lbllowcd scientific principles and rapid progress was firmly

.,, ,I2l

During the 19th century, the cham of Hippocratic tradition was broken by the germ therapy,

known as the cloctrine of specific etiology of diseaseEl. In tJre firct half of the 19'h century,

the study of drugs was included in Materia Medicatal.

Despite these prestigious glories in medicine, pharmaclnhertited from Hippocrates or Golen

and guided by ancient medicinerernained an empiric science until the end of eighteenth

century l4l. Beforc the twe[tieth ce[tury, mediciDes were maniiy consisted of herbs and

potions and it was not until the lnid nineteenth century that the Ii$t serious efforts w€re made

to isolate and puiry the active coilponents olthese rernedies. The success ofthese efforts led

to the bir I r ol rnany oI pltarm:ce'rt rcal con]panies.

I.1.2 MajorClasses of )r'ugs

A. Natural Products, Drrrg,s Obtained fro.m Plant and Animal Sources

Alkaloids

These are nitrogen contaidng compounds primarily obtained from plants through extraction

and purification and possess ph[rxacological activi4,. Majority of alkaloidal compounds are

basic in nature (e.g. m,rrphir.t, fto;:r opium plant); however, few Deutral amides arc also

included (e.g. colchicines, Aorn the Auturr.n crocus). A1l alkaloids end in the suffix ,.ine,,but

all the drugs that eod in "ine" are not alkaloids (e.g. Meperidine)trl.

Peptides and Polypeptides

These are ihe poly'trers of amino acids, obtained from either human or animal sources, and

are smaller than prcteins. Their ciasiilication is based on amino acid length that varies from

source to source. Natually occurring peptides have litt1e Io no oral activity and short half

lives(e.g. Somatostatin, a 14 aminoacid peptide: Glucagon, a 29 arnino acid polypeptide)t'1.

Steroids

These are chemical derivates of cyclopenteno perhydrcphenanthrene and can be obtained

from either human or animal sources(e.g. Estradial, Testosterone, Hydrocortisone)lll.

Hormones

Chemical substances that a.e formed in one organ or part of the body and carried to alother

organ or part through blood is callcd honllone. These are pri[cipally proteins or steroids a!1d

can be obtained eidrer natulally through animal soulces(e.g. drlroid hormones and

conjugateci esuogens) or slnihetically through recombinant DNA :echnology(e.g. Insulin)lrl.

Glycosides

These are organic substarces containiog a glycosidic hond between a sugar and a non sugar

(aglycone) moiety(i.e., a 1. ond b/w tie arl orneric carbon of the sugar and a hydroxyl group on

the aglycone). For glycor,ldes, oiigrn cau be piant(e.g. Digitoxin) or microbial (e.g.

Streptomycin, Doxorubicin) lr l.

Vitamins

These are organic su':stanr.:rs that are present in lbods and are essential to nomal

metabolism.

a. Lipid-solubie vitamins inci decl Retinal (A), Ercocalcil'eral (D), o-Tocopherol(E), and

phltonadione(K).

b. Water soluble vitamins included Thiamine(B1), fuboflavir(B2), Niacin(83),

Pyridoxine(86), Cyarocobaidnir(B i2), Ascorbic Acid(C), Folic Acid, Pantothenic Acid,

and Biotin(H)t11.

Polysaccharide

These are pollmers of sugars that can be obtained from either human oranimal souces.

These compounds call be used either directly (e.g. Heparin), afler pa.tial

depolymerization(e.g. Tinzapariq Enoxaparia) or after structual modification (e.g.

Sucralfate).

Antibiotics

These are the chemical substances produced by microorganisms that either suppress or kill

other microol ganisms (e.g. Penicillin, Tetracyciine, Doxorubicin)trl.

B. Syntiretic Products, Drugs Synthesized {rom Organic Compounds

1. S),nthetic products can contiLin similar spacing of functional goups but lack the general

stucture of a mturally occurr ing compound. A good example of it is peptidomimetics,

molecules with [o peptidc ]old and molecu]ar weigth less than 700, possess activity

similar to the original irept'de(e.g., Losartan is a pepidomimetic and an aogiotensin II

receptor antagonist).

2. S).nthetic products can have chemical structues closely resernbling those of active

flatural products (e.g., Hydroxlrnorphone, resembles Morphine; Ampicillin, resembles

Penicillin).

3. Synthetic products can also be a completely new product, obtained by scleering

sylthesized materiais tbr drug activity(e.g. barbiturates, antibactedal sulfonamides,

thiazide diuetics, phenothiazire antipsychtics, benzodiazepine arxiolytics;lrl.

1.1..3 Sources of Drugs

Crude drugs are meant to be the connneicial lbnns of admal or vegetable drugs as are

brought to the market and are utilized for the prepaxation of differcnt medicinal products.

Their value depends upon the presence of more or less definite chemical ulits known as

acdve constitutents.

According to their sources, drugs may be dividedinto the following groupst'l:

1. Organic

a. Frcm the animal kingdom, these include liver extract, lard, pepsin, hormones (e.g.,

insulin, thyroid exfact etc).

b. From the vegetable kingdoq these fom a large class, derived from roots, leaves,

bark, wood, llowers. seeds and the juice or exudates, e.9.,

Opium,Digitalis,Belladonnactc.

c.Frcm microorganisms, isolated liom soil (bacteria and fiu1gi) e.g. antibiotics.

2. liorganic

,1.

This includes metals. ,ralts. miDeral i'cids, non-metal!,(e.g., sulphur, iodine) etc.

Slnthetic e.g. general anaei thetics. sulphonamjdes and all altimalarials other than

quinine etc.These drugs are graciuallT replacing organic ones; thus synthetic salicylic acid

is being used inplace ofnal uai salicylic acid (derived from oil ofwinter$een)

Semis),mh€tic, e.g. Ethilrylo:stradi r1, ;viethyltestst( rone, Dilaudid, Dicodid etc.

1.1.4 Nomenclature oI' Dru gs

Practising various di1}'erent names for a single orug has developed confusing state in drug

nomenciature. In addition to lbmal chemical na'ne. a code flame is also used by the

manufacturing company. Wten the drug is found promising and is approved to app€ar in

market, non proprietary name is assigled to it Ly USAN council called as generic

name.When the drug is officially admitted into United States Phaxmacopeia, generic name

becomes official name. Official name and generic name may differ and thus in that condition

a proprietary or trade name is used by manufacturer. If a drug is manufactured by various

different companies, several proprietary names rnay get used l5l.

1.1.5 Constituents of Crudc Drugs and Agents Used in Pharmaceutical

Prepaiations

Two main consituents of crude drugs are (i) Inert e.g cellulose substances alld (ii)

Pharmaceutically active substaflces.

There are various agents that are added in actual active drug substance to give different

effects to the drugs.These are pharmaceutically active agents as emulsifuing agents, colouring

agents, sweetening agents, flavouring agents and,lor oiflmtment bascslll.

1.1.6 Different Dosage ['orms of Drugs

There are three basic dosage fomts or'drugs [6]:

Gas, Vapor rnd Other Dosage Forms

Aerosols

Spmys

Liquid Dosage Forms

Solutions (topical, systemic, epicutaleous, percutaneous, peroral, otic, ophthalmic,

parenteral, rectal, urethal, vaginal)

Collodions

Aromatic Waters

Enema

Elixir

ExtacLs

lrrigating Sloution / Douche

Liniments

Parenterai

Spirits

srup

Tinctures

Dispersions

Emulsions

Suspensiofis

Lotroos

Gek

Glycerogelatins

Magma

Cream

Ointment

Pdste

3. Solid Dosage Fonns

Capsules

Pi11s

Tablets

Suppositories

Effervesceflt Salts

hnplants or Pellets

Lozenges, Torches, or Pastilles

Powders and Granules

1.1.7 Drug Potency and Efiicacy

Potency of a drug is a relative measure that compares different doses (molar doses) of

diilerent drugs needed to produce same effect. Efficacy of a drug is measured by its

maximurn effect.

1.1.8 Stability o{Drug

There are various different ways through which the drug is decomposed but the highest

instability in drugs is associated with oxidation and h,vdrolysis.

Deterioratiofl of drug through oxidation is initiated by moiecular oxygen under mild

conditions. involves radical chain reaction. Mainly drugs like phenols and polrusaturated

compounds are highy susceptible io oxidatio[.

A large number of functional goups in drugs are susceptible to hydrolysis during storage.

D'ugs containing amides afld estem are significantly effected by acid or base catal]zed

1.1.9 Factors Effecting Stability of Drugs

It is found usual that dry pharmaceutical products have longer shelf life as compared to liquid

phamaceutical products. Thcre are [umerous factors that are responsibe for decreasing the

d1rg stability. These include storage form, storage time. storage container materiai,

physiochemical properties, tempeEture, humidity, atmosphere, light, oxgen content, salt form

and presence of impurities t8l.

1.1.10 Adverse Drug Reactions and Toxicit-v

Administration ofdrugs may lead to (a) Side Effects (b) Toxic Effects III:

Side Effects

These are undesirable effects a[d are produced even with the rherapeutic dose of a dfl]g, e.g.

a patient receiving atopine lbr ihe treatnent of peptic nlcer gets dryness of mouth as a

conlnon side effect.

Toxic trffects

These are encountercd after adminisfation ofa drug in large doses or for long periods.

Drug Toxicity in trIan

(a) Local toxicity: irritation, necrosis, thrombophlebitis.

('b) Systemic toxicity: the systemic toxic effects may be manifested in various ways;

1. Hypersensitivity. idiosyncrasy, anaphylaxis and allergy

2. The other systemic toxic effects may be grouped as follows:

i. Hemopoitic Toxicity: It includes anemia and blood dyscrasias

ii. Hepatotox.city: Alcohol, Chloroform, CCl.r and Cinchophen are dangerously

hepatotoxic drugs. Few may produce cholestatic Jaundice.

1.1.11 Uses of Drugs

On the bases of its usa!,e, drugs may be categorized as t6l:

Diagnostic Agents

Phamacodllamic Ag€nts

Therapeutic Agents

Destructive Agents

Prophylactic Agents

1.2 ,{NTIBIOTICS

Antibiotics (against life) are meiabolic products of aerobic spore forming bacteria artd fungi,

produced by various specieJ of microorganisms (bacteria, fungi, streptomyces,

actinomycetes), inhibits the growth of other types of micrcorganisms (both bacteria and

furgi) either by killing or stop!,ing their growth. Antibiotics that stop bacteria ftom growing

are bacteriostatic while those thifi cause bacterial cell death are bactericidialls. e-1 r].

1.2.1 Historical Background of Antibiotics

Bacteria lv'ore Ii6t identified in 167Cs by Van Lee U Wen Hoek, following his invention of

the microscope. However, it was not until the nileteenth century that their liDk with diseases

was appreciated. In i877, Pasreu. and Joubert discovered that anthara,\ bacilli were killed

when grown in culture in presence of certain bacteria. Similar observations by other

microbiologist 1ed Vuillemin to define antibiosis as the biological concept of survival ofthe

fittest (one organism desttoys another to preserve itself and survive). Tte fiIst natural

artibiotic ever used was cramicidinextaxted ftom soil bacteria, streptomycin. In 1910,

Ehrlich successfuily developcd the firct example of a puely synrietic antimicrobial drug. In

1929, accidential discovery of th€ antibacterial properties of penicillin by Sir Atexender

Fleming was credited with iritiation of new era, it was the birth of modem altibiotic

treatmcntlril. Chlolamphenicol and Chlortetracycdine were identified towards the end of or

soon after World War II{51

1.2.2 Classification of Antibiotic

Due to varied chemistry of antibiotics. chemical classification of antibiotics is of limited

value. Even then the structual similarities may lead to the lbllowing classification of

13

a. Micrclides (A iarge lactone ring) includes Erythromycin and Oleandomycin

b. Penicillin + Cephalospo.ii (il- iactam ring containg artibiotic)

c. Streptomycin and Dehy(,rostreptomycin (Amino sugar moieties)

d. Antifungai and (Nystatin + amphoteicins, conjugated polyene compounds)

e. Chloramphenicol

f. Tetyracycline

g. Miscelianeous

1.2.3 Classification Based on Nlechanism of Action, Site of Action and

Chemical Structure

1. Ceil membranes ilhibitors(polyuryxii), Nystatin and Amphotericin B (affecting

pemeabiiity and leading to ierkage ofintraceliuiar compounds)

2. Bacterial cell wall inhibitorc (Penicillins, Cephalosporins, Cephamycins,Vancomycin,

Bacitracin.Clotdmazole.Fluconazole and ltracanazole, Cycloserine)

3- Folic acid synthesis inhibitors (Sulphonamides and Trimethoprim)

4. Protein syrthesis inhibitorslamine cyclitesincludes aminoglycosies and Spectinomycin,

Tetracyciine, Eqtkomycin, Clillciomycin, Chloramphenicol). One q?e of agents effect

the function of 30 S or 50S ribosomal suburits to cause a rcversible inhibition ofprotein

synthesis(bacteriostatic) e.g. Cl oramphenicol, Tetracyclines, Erlthromycin,

Clinclamycein and Prcstinomycins while the othq twe binds to the 30S ribosomal

subur.it and alter protei[ sylthesis that eventually leads to the cell death i.e bacteriocidal

(amino glycosides)

5. Nucleic acid inhibitors(Nalidixic Acid, Rifampicin, inhibit RNA polynerase and

topoisomerases quinolones)

6. lnhibition of enz),me sy[thesis and reduction of dihydrofolate(Antimetabolities) which

block essential enz),rnes of tblate metabolism; e.g. trimethoprium, sulfonamides

7. Antiviral agents, several classes includes;

a. Non nucleoside reverse tnlscriplase iDhibitors

b. Nucleic acid analogs

c. InhibitoN ofother essential viral enzlirnes

1.2,4 Characteristics of Antibiotics

There are certain characteristics that need to be present in antibioticstel:

l. Antibiotics should not be toxic al1d should nol precipitate serum protein.

2. It should be stable and should be effective against pathogens.

3. Most preferably, it should be soluble in water.

4. It should not cause hernoiysis altd affect the vague sites advelseiy.

5. tt shouid not be p),rogenic and cause histamine like responses.

6. It should be well tolerated in required the dose and have few undesireable side effects.

1.2.5 UIa.ior Classes of Antibiotics in Clinical Use

The major antibacterial drug curently in use may be categorized in multiple ways, two of

these impotant ways are: (i) by economic impact and (ii) by bacterial diseases they are

prescribed to treat. On the basis of first category, antibiotics are classed as cephalosporins,

macrolides, beta- lactamase inltibitors, penicillins, and quinolones. On the basis of secrnd

categoey, antibiotics are classed as cephalosporins, penicillins, fluoroquinolones, macrolides,

tetacycli[es, aminogiycosides. glycopeptides and all other systemic antibiotics 110].

1.2.6 Spectrum of Activity and Nlechanism of Action

Few antibiotics that have ability to antagonize the gowth of numerous pathogens have

eamed the title 'broad spectrurt" illl. Observatiors reveal that mechanism of action of

antibacterial drugs is related to effecting fie bacterial grcMh, eithff by slowing down the

growth (tracteriostatic) o. killing the bacteria (bactericidal). Studies related to mechanism of

action shorved that there are four nrajor targets in bacterial pathogens: cell wall biosynthesis,

protein biosynthesis, DNA replication and repairi and folate coenzl,rne biosynthesis tl0l.

1.2.7 Chloramphenicol

It was the first levorotatory aromatic nito group containilg broad spectrum antibiotic

introduced in medicinal ,sei"l. It *us fimt jsolated by paul Buskhalder in 1947 Aom

Sheptomyces: S. venezuiae, S. omiyamensis and S. phacocbromogenes. named as

Cl' oromycetin due to its chlorine contmt and because it obtained from an actinomycete. It

was first prepared synthetically in 19.19t1,1r1. It is effective against both viral and bacteriai

infectionslal.

1.2.7.1 Structure of Chloramphenicol

This antibiotic, CrLHrrOsN:Cl:,2.2-dichloro-1r'-l(lR,2R)-2-hydroxy-l-(hydroxymethyl)-2-

(4-nitophenyl)ethyl]acetamideis unique among natulal compounds in that it contains a

nitrobenzene moiety and is a de.ivative of dichloroacetic acidl5l.

1.2,7.2 Nomenclature of Chloramphenicol

Cldoramphenicol possessess two chiral carbon atoms in the acyl amidopropanediol chain that

ieads to four stereoisomers. Out of six possible isomers in it, or y the natulally occurrhg

form is biologically active. Biological activity is associated with D-threo isomell I'121.

1.2.7.3 Antibacterial Spectrum

Chioramphenicol exhibits wide spectrum oiantimicrobial activity. Pimadlary it is

bacteriostatic but may be bactedocidal ior cenain speciest5l.

1.2.7..i ivlechanism of Action

It is capable ofpenetrating right into fie centEl nervous system so is an altemative therapy

for meningitis. The metabolism ofChloramphenicol involves. fomation of3-o-glucuronide,

16

rcduction ofdre inherent para nitio moiety to amine, hydrolysis ofamide group, hydrolysis of

alpha chloro acetamide and reduction producing alpha hydrooxyacetyl analogue.

Cbloamphenicol inhibits protein s)'nthesis by penetrating into bacteial and eukaryotic cells

through diffusion and binds reversibly to 50 S ribosomal subunit t5l.

1.2.7.5 Adverse Effects and Degraded Products

There are various ontoward effects that are associated with chloramphenicol arise due to the

fact at dudng the inhibition of synthesis of enz;,rnes of mitochondriai membrane by

inhibiting ribosomal peptide tanslemse, it also effects c),rochrome oxidase, ATPase and

lerochelatase (lhe finai enz),rne il1 heme bioslrthesis). These adverse effects geflerate

hlpersensitivity reactions, hematological toxicity thar involves bone marrow. Other than

allergic and toxic eilecis, it aiso disturbs normal microflora ofthe body that leads to adverse

reaction in biological seme i5l. It is also associated with gastrotestinal distubalces aIrd

toxicity in newboms infants Ir3l.

1.2.7.6 Clinical Uses

It is an impotant drug for the heatment of tlphoid fever, bacterial meningitis, anaerobic

infectiofls, Rickettsial diseases, brucellosis, lar)'ngotracheitis or pneumonia caused by p-

Iactamase producing strains, H. influenza meningitis, urinzEy tract infectiofls and systernatic

salmonella infections l5l. It is used for the treatmert of eye infection because of its wide

antibacterial spectrum and capability to penetrate in ocular dssues and the aqueous humor.

However it is ineffective in cl iunydial eye inf'ections lr3l.

1.2.7.7 Structure Activity Relationship of Chloramphenicol

In propane diol moiety, stereochernical substituents upon the asymmetric carbon atoms I and

2 are absoluteiy essential ibr aotimicrobial activiSrzl.

17

p-nitro pirenyl Group

The antibacterial pote[cy is propotional io the rclative e]ectronegatiyity of the pala

substituem ofdre phenyi goup. It appears thaa p-nitlophenyi goup may be replaced by other

aryl gioup without appreciable loss in activitfll-r21.

Nitro Group

Nitrogroup may be replaced by a vaiety of eiectronegative groups without drastic loss in

antimicrcbial activity but would genemte less active structure than chloramphenicol.

Halogen

2-NHCOCFI(fluoro) derivative is 1.7 times as active as chioramphenicol..

Hydroxyl Group

Conversion ofalcoholic group on C-1 to l(eto causes appreciable loss in activity.

Dicholoracetamido SideChaiir

If the dichloroactamido side chain is deleted, rest of the sttucture of chloramphenicol has

or y 2yo of activity of the intact antibiotic molecule. This group may be changed with the

rcstriction that strong electronegative chamcter in the acyl residue should maintain and the

size ofacetyl group is not exceediflg critically.

1.3 CORTTCOSTEROID

1.3.1 Steroids

Steroids are members of a large class of lipid compounds called terpenes, biogenically

derived from the same parent conlpound, isoprene, C5H3, shate the same basic fully satuated

(perhydrc) threc six carbon atoms fused rings structure (together the phenatherene part),

fused to one five- carbon atom .ing and coliectively known as cyclopentaphenantherene.

These are found in a variety of diflerent marine, tenestriai and s),nthetic souces arld are

chemical messengem, Laown as honnones. Due to their nonpolar chamcter, thcse may cross

cell membrane. So stercid may leave the cell in which it is s)nthesized (glands) and enter the

target cell thrcugh blood strean.The vast diversity of the natural and synthetic members of

this class depends on variations in side chain substitution (p.imarily at C 17), degree of

unsatuatioD, degree and nature of oxidatio[ and the stereo chemical relationships at the ring

junction. Those stercids having alcoholic hydroxyl attached to the ring system are known as

sterois l14_l?1.

1.3.2 Structure, Numbering, Nomenclature and Stereochemistry of

Steroid

The four rings are lettered A, B, C and D and carbon atoms are numbered beginning from

ringA. Steroidal ring B,C and D are always fused trans while ring A and B may be linked

tmfls or cis. In most naturally occuriog steroids, ring A and B are fused trans. All stsroids

possess at least 17 carbons. Nlany steroids have methyl goups attached at C-Io(position 18)

ard C-l3(position 19), called as angular methyl groups. Instead of angular methyl goup,

hydrogen call be lbund at C-13 (position 19) iDld is not substituted when ring A is aromatic.

Positioll 17 can be substituted. unsubstituted, and,' or oxygenated. Usually a side chain is

attached to C-17 and related series of steroids are named after their furdamental ring systems

1g

i.e. determinethe root name. Substituents that extend below the plane of the steroid are

reffercd as a (designated by broken line) and those extend above the plafle arel (designated

by bold/ soiid line). By convention the angular methyl goups (numbered 18 and 19) are

written as being above the ring systerni.e. in B configuration. Steroidal nomenclature involves

a wider use of (R) -(S) system for desigmting the stereochemistry in the side chain. In

steroidal side chain, stereogelic centers are aiso denoted preferentially with the R and S

nomenclature. Steroid sketelon shows a specific stereochemistry. All thee of each six

membercd rings can adopt satrain free chain colformations. Unlike simple cyclohexane

rings, large rigid molecules carnot undergo ring flips i.e. chain- chain interconveEions.

Substituents on the steroid .ing system may be either axial or equatorial but adopt more stable

equatorial substitution due to steric reasons[14-r6].

1.3.3 Types of Steroids

On the basis of fte physiological fuictions. stercids can be categorized as followslra r?l:

Corticosteroids(Glucocorticoids and Niineralocoids)

Glucocofiicoids are a class of corticosteroidal hormones chamcterized by arr ability to bind

with the cortisol receptor and trigger similar effects. Due to its ability to regulate many

aspects of metaboiism and immune lirnctions, glucocodicoids are often presc.ibed as a

remedy for inflammatory conditions such as asthama and arthritis, example: cortisol

Ntinerulocorticoids are coticosteroids thathelps to maintain blood volume and corltrol .enal

exqetion of electrolltes. Exampie: Aldosterore

Anabolic Steroids (Anabolic Androgenic Steroids)

These are a class of l1aturai and s]mthetic steroids that promote cell growth and division by

intencting with a[drogen receptors. resulting ill growth ofseveral t]pes oftissues, especially

muscle and bone. Examples: Testosterone, Nandrolone and Methandrostenolone.

Sex Steroid and Conadal Steroid

These are a subset of sex hoflnones that interacts with androgen or oesftogen receptors of

vcrtebrates to prcduce sex differences(primary and secondary sex characters) and support

reproductiol. These include a[drogens, oestrogens and progestagens. Examples:

Testostercne, Oestradiol and Progest$one.

Androgens are i9 carbon steroids that contain the basic perhydro- 1,2_

cyciopentaphenantherene ring system with the C- I 8 and C- I 9 angular methyl goups attached

to it. A primary function of it is to maintain the male sex organs and secondary sex

characteristics.

Estrogens are characterized by having au aromatic A-dng and thus a phenolic character.

Esffogens stimulate the gowth and developmeot of the fernaie reproductive organs and the

secondary sex characteristics. Another pdmary function of estrogem along with progesterone

is to regulate the o\,llatory cycle.

Prcgesterones are the principal progcstin in mammals secteted primarily by the corpus

luteum ofthe ovary. lvlain responsibility ofprogesrerone, together with eshogen, is to preparc

the endometrium for pregnancy.

Ergosterols

These are steroids that occur in fungi, and ioclude some vitarnin D supplements.

The term vitamin D refers to a group of seco-steroids that possess a common conjugated

t.iene system of double bonds. Vitamin D3 and D2 are the best known examples. Primarily,

vitamin D3 is found in vefleberates, whereas vitamin D2 is iound in plants.

Phytosterols and Plant Sterols

These are alcoholic steroids that naturally occur in plants. Examples: p_ sitosterol

Similar to Cholesterol, plant sterols have a stmctual and fuctional role in biological systems

and serve as intemediate in the biosenthesis ofan assortment ofbiologically active ste.oids.

1.3..1 Adrelal Cortex Horr ones

'Ihe hormones elaborated by the adrenal cortex aie stercidal derivates of

cyciopentaperhydrophenantherene related to the sex honuones. At least 50 adrenocortical

hormones are produced by the adrenal conex. Chloestrol may be considered as the starting

poi11l tr5 l7l.

1.3.5 Types ofAdrenal Cortex Steroid and Classification of Steroid

The adrenai cortex synthesizes two classes ofsteroids: The C- 21 steroids derived the adrenal

cortex arld their metabolites are collectively called as corticosteroid and C-19 steroid as

aadrogensl5'?1.

1.3.6 Corticosteroid

It is a biosflthetic precu$or to aldosterone and is an intermediate that has weak properties of

borh previouslymentioned gtoupstl5'71.

1.3.6.1 Classification of Corticosteroid

It is traditionally classified into three goups: glucocorticoid, mineralocorticoid and cortisone.

All three have few featues in common; Hydroxy methyl ketone group at C-17, the C- 1l

hydroxyl functional group ard o B urNaturated ketone system from C-3 though C-5.

Glucocorticoid

These are thc adreDdl homoncs involved in the control of glucose. Promotion of

glucogenesis iiom amino acids is one of the functions of steroid. This class possess an O or

OH substituent at C-l1 (Corticosterone) and an OH group at C-17 (Cortisone and Cortisol),

€xerts its action on organic metabolism. In human the chief giucocorticoid is cotisol.

Nlineralocorticoid

Those stercids whose main function is the control of eiectreol),te balances are known as

mineraloconicoid. These steroids are responsible lor Na retention and promoting rcnal

excretion oi I( This laci(s the oxygenated goup ar C-17 (deoxycorticosterone and

aldosterone), act prirDarily on elecfoi]4e and watff metabolism. In human chief

mineraiocorticoid is aldosterone.

1.3.6.2 Structure, Numbering, Nomenclature and Stereochemistry of

Corticosteroid

In formal chernical nomenclature. the adrenocorticai hormones are described as de.ivates of

andosterone or ofpregnane. Double bonds are indicated blthe slmbol A with superscriprs to

ildicate the position ot'the double bond, e.g. cortisol is designated as 1 I p,17o,21-trihydroxy-

14-pregrene- j.2u-dione l' .

1.3.6.3 Nlechanism of Action of Corticosteroid

Corticosteroids act by controlling the €te of synthesis ofproteins. The corticostsroids react

with receptor proteinsin the c),toplasm of sensitive cells to form a ste.oid receptor complex.

This complex undergoes a modification, moves into the nucleus, where it binds to chromatin.

Informatioo carried by the stercid or more likely by the receptor protein directs the genetic

apparatus to transcribe R.\At5l.The most impotant mineralocorticoid, aldosterone is an

aldehyde as well as ketone; which regulates the reabsorption of sodium and chloride ions in

the kidney; and increases the loss of K ions. Cotisol or hydrocortisone. the most important

glucococolticoid has the function of ir1creasing glucose al1d glvcogm concentrations in the

body. These reactiol1s arc complered in the liver by taking fatty acids from lipid storage cells

and amino acids from body protein to make glucose and glycogenllal

1.3.6.4 Structure Actiyity Relationship of Corticosteroid

An oxygen function at C-l1 is oot an absolute rcquirement for anti_itflammatory action ifother activating feactures like l-2 double bond and 6 a- CH3 is present coupled with 16 a_

OH. wl]en C-11 hydroxy is present. however its stereochemistry s[ongly effects the activity

ofcompoundlr5J.

Reduction of the C-20 ketore g.oup to a C-20 hydroxyl configuation lelds a substance

having little, if any, biological activity. Corticosteroids with a hydroxyl goup at C-17

undergo an oxidation that yields 17 keto steroids and a two - carboll fragnent. These 17-

ketosteroids are totally lacking in corticosteroid activity but, in a ferw instances have weak

andrgenic propetiest5l.

Chaages in molecuiar structure results in aiterations in absorption, protein binding rate of

metabolic kansfomation, rate of excretion, ability to transverse membranes, and intrinsic

effectiveness of dre molecule at its site of action and thus may bring about changes in

biological potency.

Strucure Activity Relationship of Adrenocorticosteroid

Ring A

The AC-4 double bond and the C-3 ketone are both necessary for typical adrenocorticoid

activity. Intoduction of a 1,2 double bond as in Prednisone or Predinisolone, eDhances the

mtio of carbohydrate-regulating potency to sodium retaining potency by selectively

increasing the {bnner. In addition, prednisolone is metabolized more slowly than cortisol.

Ring B

6 a - substitution has unp,-edictable effects. In the padicular instance of cortisol, 6q-

methylation iflcreases anti-irlflammtory, nitrogen-wasting and sodium- retaining effects in

man. In coltrast, 6q- methyl r.rednisolone has slightiy geater anti-inflammatory potflcy and

fewer electrol)tes - regulating potency than prednisolone. Fluorination in the 9a position

enhances al1 biological activities of the codicosteroids, apparently by its elechoo

witfidrawing effect on the 11p-hyd.oxy goup.

Ring C

The presence ofan oxygen fimction at C-l I is indispensible for significant anti-inflammatory

and carbohydrate regulating potency, as demonstrated by desoxycorticosterone.

Ring D

16- Methylation or hydroxylation eiiminates the sodium retainirg property but only slightly

modifies potency with respect to effects on metabolism and inflarffnation.

Al1 presently uscd anti- inflammatory steroids are 17o- hydroxyl compounds. All natural

coriicosteroids and most of the active synthetic analogs have a 2l-hydroxy group. Though

some glycogenic and antiinflammtory activities may occur in its absence, ils presence is

required lor sigrificant 'odium- retdrning acti\ ity.

1.3.6.5 Uses of Corticosteroid

Corticosteroids axe the most efficacious treatment of asdrna, ard inhaled corticosteroids are

considered to be a fiIst line therapy for asthma. Nasal spray topical corticosteroids are widely

regarded as the reference standard in rhinilis theapy.

Particularly glucocorticosteroids are highly effective agents for the heahnent of chronic

inflammation. Hematologic diseases are also teated with glucocorticoids. These include

2\

idiopathic and acqui.ed hemol)'tic anemia and even adrenal hlperplasia may respond to

steroid therapy. Among drugs useful in the treatment of leukernias and llmphomas,

corticosteroids are in the fbrcftont.

Table I

Phannaceutical Drugs Containing Chloramphenicol

ChloramphenicolS.# Drugs'Name Composition of Chlor.mphedcol Dosage Form Compatry

Chlorophenicol i % w

ChloromaxChloromedChloramphenicol 40mg/mlCirloramph€nicol,+omg/rnl-Chlora-x

Pharm€dicNiedicrafiBoschLCPWElite PharmaPlivaLCPWGeoftnanPDHRemhgtonSanta

Dosaco

LCPWPolyhneTabrosNabiqasimErcsPizerPlivahzaLCPWLiskoEpochUnexoCeiremSharex

DosacolrzaPharma WiseNabiqasimEpochErosPDHBarert Hodgson

DosacohzaPharma WiseG€oftltanIonvotekMacquinsMedipakFarmigeaOphth PhamaZafaReminglonSanta

40 mg/rnL40mg/ml

InjectionInjectionInjectionInjectionEye OintmentEye OintmentEye OintmentEye ointmenrEye OiDtmentOiniOphth ointSuspcilsionSuspensioDSuspensionSuspensionSuspansionS),rupDropsDropsCapsulesCapsulesCapsulesCapslrlesCapsulesCapsulesCapsulesCapsulesEar DropsEar DrcpsEar DropsEar DropsEar DropsEar DropsEar DropsEar DropsEar DropsEye DropsEye DropsEye DropsEye DropsEye DropsEye D.opsEye DropsEye DropsEye DropsEye dropsEye dropsEye drcpsEar DropsEye drops

ChloramphenicolGeoclorNeo- PhenicolOptachlorSantochlorCt oramphenicolCl oramphenicolCh loramphenlcolClrloramphenicoLChloramphenicolChloramphenicolComycetinErophenicoiChloromycetinClrloramphenicolChloramphenicolChloramphenicolChloramphenicolEpophedcol

ChlorogenBiomycineChloramphenicolCi oriunphenicolChloramphenicolCbloramphenicolComycetinEpophenicolEmphenicolNeo-PhenicolChloropticCl oramphenicolChloramphenicolChioramphenicoiChloramphenicolceochlorInvochlorManophenicolMedichlorOcuchlorOphth- ChlororbachlorRemicolSanrochlor

,X*r*1%w/wI o/o wlw1%\v/w

0.5 % v0.5 % w/v250 mg250 mg250 mg250 mg250 mg250 mg250 mg250 mgl%wlv

l?owlvlo/. w/'rln/o w/vl%oNlv0.5% w/v

o.tr" *r,0.5% Wv1% w/v03%wlv0.5% wlv0.5% wv0.5% wivlo/. wt',0.5% wvi0.

S.# Drugs'Name Compositiop of C[loramphetricol Dosage Form gqoptuyEye drops51. Schazomycetin 0.5% w/v

52. Spersinicol o.sYowlv0.5% wv

56. Virachlor 0.5% dv57. Erophenicol 0.5% w7v

55. VI-Chlor

0.5% Wv0.5% v

60. Optachlor 0.5% wiv

Eye drops NovartisEye drops VegaEye drops vegaEye d.ops Jaens

Eye drops MedipakDrops ErosDrops HelixDrops RexDrops Remin8on

54. VegacNor 0.5% w/v0.50/0 v

58. Feooptic59. Oricol

Table 2

Pharmaceutical Drugs Containing Prednisolone Acetate

Predlisolode AcetateS-# Drugs'Name Composition ofPrednisolo[e Acetate Dosage Form Company1. Fortipred o.|Y.wlv2. Lophase 0.1Yo wlv3. Mildopred 0.l Yowlv4. Ocupred-Mild 0.1 % dv5. Ophtha-Pred 0.1% v6. Optopred 1.1 Y" wlv7. Pollpred 0.1 % Wv8. Pred+ a.l ok wlv9. Pred-Forte 0.1 % v10. Pre&ipos 0.5% w/v11. Prednirex 0.1% v12. Prens 0.1% v13. Prednitek 0-1% v

Eye Drops RemingtonEye Drops EpochEye drops ReminglonEye Drops FormigeaEye Drops Ophth PharrnaEye Drops SanteEye drops PolyfineEye Drops SchazooEye Drops Barrette HodgsonEye Drops UrsaphamE),e Drops RexEye Drops VegaDmps lonvotek

Table 3

Phanaaceutical Drugs Contairirg Dexamethasole

DexamethasotreS,# Drugs'Name Composition ofDexam€thasone Dosage Form Companyi. Aldron2. Dexacort3. Dexadron4. Dexamethasone5. Dexamethasone6. Dexamethasone7. Dexamethasone8. Dexameihasone9- Dexamethasone10. Dexamethasone11. Dexamethasone12. Dexamethasone13. Dexamethasone14. Dexamethasone15. Dexone16. Iladexon17. Hamazone18. kzamedusone19. Omdex20. Phesore21. Dexamethasone22. Dexamethasone23. Dexamethasone24. Dexamethasorc25. Dexamethasone26. Dexamethasone27. Dexamethason€28. Dexamethasone29. Dexamethasone30. Dexoptic31. Mexidex32. Medidex33. Methadex34. Ophth-Dex35. Optadex36. Spersadex37. Steridex38. Binadex

-19. Dexamex,+0. Metaarex

Tabiet'I abletTabIetTabletTabletTabletTabletTabletTabletTabletTabletTabletTabletTableiTablelTabletTableiTabletTabletTabIetInjectionlnjecrionlnjecrionInjecrionlnjecdonInjectionInjectionLrjectionIDjecuonDropsDropsDropsDropsDropsDrcpsDropsDropsEye DropsEye DropsEye Drops

HarmarmPrcgressiveEpoch

ArdinGabaGeofnnnLiskoMunawar PharrnaR€gentTabrosUnexo

XenoIrPharrna WiseAifaiah PharmaHall]azbzaOBS/OryanonPhannacareAmrcsElko

PlivaS.J&GFauzulEllaheS.J&GFauzulEllaheTabrcsUnexo

SanteAlconMedipakJaens

Ophth PharmaRemingtonNovartisKobecBarrett HadogsonBoschRex

0.5mg0.5m9

9'-t

.

-

0.5mg0.5m90.5mg0.5mg0.5mg

3"'

O.t *r,,0.1% v0.1o/., /v0.1Yo wl\0.1% v0.1Y"wl,r0.). a/" ' h0.1% v0.1 % w/v0.1 % v0.1 o/o ttt/v

Table 4

Pharaaceutical Drugs Containing Chloramphenicol, Prednisolone Acetate andDexamethasone in Combinatiol

Chloramphenicol + Pred[isolone AcetateS,# Drugs'Name Compositiou Dosage Form Compstrvl. Prednisynth 0.5% CtrJoramphenicol+ 0.2 oZ Prednisolone

Acetate2. Prednicol 0.5% Chloramphenicol+ 0.2 % Predrusoione

Acetate3. Predni-C

Eye Drops

Eye Drops

Eye Drops

Eye Drops

Schazoo

Remington

Mediceena

Formigea

0.5% Chloramphenicol+ 0.2 9/o Prednisolone

4. Occupred- 0.596 Chloramphenicol+ 0.2 % PrednisoloneChlor

Chloramphenicol + D€xam€thasoneS.# Drugs'Nam€ Composition Dosagetrorm CoEpaoy1. Decachlor

2. Dosachlor

3. Dexoptic-C

0.5% Chlordmphenicol+ 0.1 %Dexamethasone0.5% Chloramphenicoli 0.1 %Dexamethasorc0.5% Clrlommphenicol- 0.1 %Dexamethasone

Drops

Drops

Drops

Drops

Sharex

Dosaco

Sante

Farmila4. Fluorobioptal 0.5% Chloramphenicol+o.2 %Dexamethasone

2.0 RESijLTS AND DISCUSSION

2.1 DEVELOPN,IENT OF ULTTR.A FAST HPLC ANDTLC DEIISITOIvIETRY METHODS FORDETER\IINATI()N OF CORTICOSTEROID ANDANTIBIOTIC IN PHAfuMACEUTICI\L PRODUCTS

Validate<i Ultra Fast HPLC Methods for Determination of

Corticosteroid and Antibiotic in Pharmaceutical Produots

34

2.I.l.l Prednisolone Acctate and Chloramphcnicol

A number of corlicosteroid and antibiotic combinations are lrcqucntly uscd as anlibacterial

agents to cure infections associated with eye. These oonbinations are available in diaferent

dosage lomrs including eye ointmcnt and ophthaln'lic sUspcnsions{r31. Prerlnisolone Acctate,

11il, 17-dihydroxy-3, 20-dioxopregna- 1,4-dicnc,21-yl acctatc is uscd in polychemotherapy ol

cancer and as ao inmunosuppressive to treat allergic disorders and hypersensiti!iry

rcactionst'e "1. Chloramphenicol, a phenicoiic antibietic,2,2 dichloro:1r'-[( lR,2R)-2 hydroxy

I -(hydroxyrnethyl)-2-(4 niirophenyl)ethyllaceiamide has widc spcotrlurl antibacterial activity

and is used for troat,nenl o I ricketts ial d iseases, chlarnydial diseascs, gram t!eandgram vc

bacterial infections and topically for curing superficial coniunctivial infcctions 0rl. Many

analytical methods are found reporled for detormiratior of Prednisolone Acelale in human

serum, urine and pharn'raceutical preparalions t24-rrl. HPLC mcthods are also lound rcported

for determination of Chloftunphenicol ;n conrcdo crcaft, blood, gastric contents, urine,

phannaceutical fonnuiations, tissues and CSf I'o-'ul. A large number oI LIPLC mcthods are

Iound reported, for dcterminalion of Chloramphenicol along with corticosteroids, including

rlydrocorlisonc Acetate, Dexamethasone and Doxamethasone Sodium phosphate in djlv

auristilla, lufuye, phannaceutical forrnulations, ludi crcanrs, cosrnetics, kangyan paint and

collyria products I" ttl. HPLC nrcthods are also founct reporrcd fir simuttancous

dotcrmination of PrcdDisolone Acetale and Chloranlphenicol in phalgtaceLrticai prcparations

['65'1. S"purut" assay rnetho<1 is found reported in USP 2008 Ib. rhe deternrinalion of

It(rdnisolono Acetate and Chloramphenicol in Ophthalmic OintDcnl.

Many Uhra Fast HI,LC methods arc found reported for determination oi prednisolone

,4cetate in cosmetics and Chinese medicinal preparations [53 601. Ultra Fast HPLC mcthods are

also found reportcd fbr detcrmination of Chloramphenicol in aquaric prodLrcrs, in

pharnaccutical prcparations and in cosmetics tu' utl. No UltLa Fast HPLC mclhod has bccn

found reported so far ibr the 6l ralysis of prednisolone Acetate and Chlonmphenicol in

combi[atio[ in phanlaceutical pro('ucts.

A simple and validated Ultr.a Fast HpLC method was developed for determination ofprednisolone Acetato atd C5ioialitphenicolin pharrnaceutical preparations. The proposed

Ultla Fast IIPLC melhod was or-veloped on Zorbex Eclipse XDB Rp-Clg column (50 x 4.6

mm I.D., 1.8 pm) anal,zing bo.h the acrive componenrs within I minute, in run time of 3.5

minutes using lineai gradient mob:le phase composed of acetonitrile and water with ljltra

Fast HPLC paramet:rs including flow rare of O.g mvmin. 1 pl injection volume al1d

photodiode array detection and ELSI] a! muiti wavelengths of243nm and 27grun. Following

the recommended IC)-. guidelines. th-, developed metliod was validated by performing

linearity, accuacy; inter day and intraday precisiot, specificity, robustness, LOD and LOe

parametem. The response was linear o./e- the concentration range of 50_1000 pgln , with

corelation coefficient of0.99961and 0.99919 for Chloramphenicol and prednisolone Acetate

respectively (Figurc 2 and 3). Method was lburd reliable fbr quality control laboratory

anal)sis and was successfully applied for detection of eleven phamaceutical products

including five eye drops of Prednisolone Acetate,two eye drops of Chloramphenicol,

capsuleand ointment; and two eye drops containing Prcdnisolone Acetateand

Chloramphenicol in combination.

Optimization of Ultra Fast HPLC Nlethod

With a view to deveiop reliable and rapid method, procedure was optimized considering the

injection volume, mobile phase flow rate. column temperaturc and wavelength. Different

solvent systems were tested in different gadient ratios including methanol: water, methanol:

0.5 ru\4 sodium acetate. acetonitrile:0.5 mM sodium acetate. acetonitrile: water and

acetonit ile: 0.1 % lormic acid. Methanol : water and methanol : 0.5 mM sodium acetate

gradients were producng very higl pressure while mobile phase with acetonitrile and 0.1 o/o

formic acid was given peak I plitting. Though mobile phices having 0.5 mM sodium acetate :

acetonitdle were giving \,rrry sh;,ry peaks but due to th,] prcblem of producing large number

ofbubbles, it was rLor se.te*ed as the finai mobile phase;. Almost forty gradient systems were

checked aDd llnal selectioii ,vas done lor the mobile pilnse having composition of acetonitrile

and water. The pei.k a, liof 0.648 t0.0133 was recogrized as Clrioramphenicol while the

peak at Xr0.888636 10.0r)9',1 was recognizeC as prednisolone Acetate in comparison to the

individual ch orratograms cbtai,eci (Figue 4). The o'timar DAD murtiple wavelengths at

243ru1 Q" ma,x (,f Pred[ir]oion€ Acetate) and 27gnr.r (1, max of Chloramphenic.l) for

derecti,x were selectecl aftor nu1ni1lg tire chromatogram at five different wavelengths (243,

278,250,260,254). 254tun was giving the highest absorbancr: after the l" max of these active

compon{rnts. Deteclion at 260 ,,,, showed equal concentlation for both prednisolone Acetate

and Chlora,mphenicrl with i00% recovery of both the components but was lesser than 254

nm while 250nm uas showinl; 1ow absorbance than both 254nm and 260 nm, so final

selection was done lbr i rnar of both the active components. Injection volume of lpl was

selected after applying 3pl a1ld 6Ii idections. Column temperatue of 25"C and 30"C was

checked but it was found that ru] at 25'C increased the retention time.

Predn'solone acetEiQ

100

o co..et€tion: o.99999

Figure 2

Method

o

Figure 3

Calibration Curveof Standard Chloramphenicolfor Ultra Fast HPLC Method

-JI

I.ooll5lo ll

1)

t

l"mal 243 rur Predni

nl,qu

120

100

80

60

40

20

o

o2R€teotion Time (n{n)

Figure 4

Chomatogam of Standard Chlommphenicol ard Prednisolone Acetate in

Combination Simultaneouslv Detected at Lambda Max of243 nm and

aqP6ll-ll

il

:l:ll

I

-lI:l-lIJ

-]-t-o

MAU120

100

80

6J

4A

20

o

278 nm Respectivelv through DAD Detector

Figure 5

ChromatoqramsofPharmaceutical Ploducts ContaininqPrednisolone Acetateand

Chloramphenicol in Combination alone\.ith Standards

t

Figure 6

Chromatogram of Prednisolone Acetate Pharmaceutical Products alongwith

Standaxds

4t

Figure 7

Standards

4i

Nlethod Validatiorr

Lineariqt

The standard calib.aticn curves in corc(ntration ranges of 50-1000pg/ml for both

Prednisolone Acetate urntl chioramphenicol were found linear with correlation coeflicient of

0.99999 and 0.99961 nrsttectively. Linear r,:grission data of the calibration curves from

triplicate injections of ser,en standard wor.dng solutions were used to evaluate linearity,

Table 6.

Pre L'is io n and Ac c uracy

Inter-oay and iotra day coetficients of variation werc observed at three concfitration levels

(100, 200,4C0 gg/ml). The accuracy was above 94 yo for inter-day and 93% for intra day

precision. The %RSD is fountl to be less than 2 0/o in all inter day and inhaday results.The

repeatabiiity ofsample applicahon and measurement ofpeak arca were expressed in terms of

0% RSD and the results are summarized in Table 7.

Linit of Detection snd Limit ol Quna rtcadon

For Prednjsolone Acetate, LOD is tbund to be 6.689 ng/pl ard LOQ was 20.27 ng/pl while

lbr Chloramphenicol, LOD is found to be 36.893 ng/pl ard LOQ was 111.79nglpl. Results of

validation parameters are summarized in Table 8.

Robustness

Selected parameteN (flow rate. column temperatue. and wavelength) of proposed method

was var-ied to 1 5olo variatioD at three levels. Replicate iDjections (n = 3) of standard solution

at tkee concentmtion levels were perfonned. SD for peak arca was calculated and is reported

in Table 9 as %RSD.

Spectrtctry

Peak purity for both Prednisolone Acetate and Chloramphcnicol was analyzed by cornFaring

the chromatograms of standard and pharmaceutical products. Peak apex, peak slarl and pcak

cnd were taken undcr considcration. No other peak was ellrting at .nr of Pred n iso lone ,A.cetate

and Chloramphcnicol.

Recover! studics

fte - analyzcd pharnlaceutical products were checkcd for rccovery studies with standad,

added up to 25 %, 50 % and 75 % ofdetected concertration and results are colnpilcd in Tablc

l0- 12.

Analysis of Pharmacculical Products

tJllra last IIPLC nlelhod al DlLrltiplc wavelcnglh for determination of Prednisolone ,^celale

and ChlorarnphenicoL using ELSD and photodiode detector in eleven phannaceutical

products is developed lor the first time (Figure 5-7). Two peaks at -&' of 0.648 1 0.0133 and &

0.888636 10.00971 were identified in chromatogram as Chloramphenicol and Prednisolone

Acetate in conrparison to sta[da1d chromatogram. No excipient inleLference was observed in

Chlolaraphcnicol and Prednisolone Acetate indiv;dual pharmaccutical products. ln

PrcdnisoloDe Acetate ophthalmic slrspcnsion drug cooterts wcrc lound to be 85.5 %, in l,red

r,86.5 % in Prens, 91.6 % in Ophthapled, 98.7 % in Mildopred and 95.4 % in Predfo e. For

Chloramphenicol eye drops, drlrg content was found to 9i.9 % in Spers,nicol,98.6 % in

Chloroptic and 102.2 % in Chloronrycetin capsule wlrile 98.8 % ir Oplachlor ointment. In

combined pharmaceutical products of Prednisolone Acetate and Chloramphenicol, drug

contcnts wcre found to be 105.3 % and 89.9 yo and 89.6 % artl97.i% for Predoisylllh and

Prednicol rcspectively. Low RSD% .eflecled the suitability of the devcloped Ultra Fast

HPLC nethod lor routire a ulysis of Prednisolone Acetate and Chloramphenicol in

phamaceutical products. Resull i are summa zedinTable 13.

'fable 5

Gradient Program of Ultra Fast HPLr-- Method for Determination ofprednisoloneAcetate and Chloramphenicol

S.# Time in minutes Sc[vent A Solr"nt B Florn .ut"--G.adi"it

(llilliQ Acetonitrile mvmin

Nater)

10 42 58 0.8 Linear

2t 1?. 58 0.8 Linear

32 12 88 0.8 Linear

4 3.5 12 58 0.8 Linear

Table 6

Linear Regression Data ofPrednir rolone Acetate anc. Chloramphenicol for Ultra FastHPLC Method

x, Ltr"- .7--1ffiRange

pg/mL

Equation ng'pL, r'g/y,L

Prednisolone Acet:rte

0.8886 i 50-r0OO 0.99999 Y= 7.387 x 1.750ri 7:S7iTO-O-OSS 20271

0.0097 10r+

1.7506

Chloramphenicol

0.648 50-1000 0.9996 Y=3.112x 8.279'7 3.712i io-36-89 111J9

10'r'.8+ 0.0133- .279',;

Table 7

Aralysis for Repeatabih ty and Intermediate precision of prednisolone Acetate

Cbloiamphenicol (n = 3 ftr.- interday ard 6 for intraday)

and

Prednisol( ne Ac(.tate Chloramphenicol

I4Es&yjlrgs!! ion lntradav Precision

Conc

'lgtLL

RSr,^ o/, Accuracy Conc

ngpL

IDterdav Precision

RSD % Accuracy yo

Interdav Pr€cision

Conc

nC/pL

RSD % Accuracy Conc

?/o ng/FL

RSD % Accuracy 7o

]100

1,.,,

]ro

l,*.,

200

600

o.'t49 t02.4

1.160 101.3

0.770 100.2

0.561 98.8

1.040 99.9

1.471 gr) g

100

200

600

1000

100

200

600

1000

0.345 98.2

0.817 97.2

1.288 98.9

1.987 98.8

1.998 102.0

0.207 101.1

1.112 101.0

0.83.1 99.5

lable 8

Sunmary of Validation Paraneters

Prednisolonc Acelate Chtorarnphcnicot

l.iD(:rrir5 rxngc 50i Lr00 ng pJ -- rg ,.1

Corrclatiotrcoelficicn{ 0.99999 0.9996

Limit ofd€tcction 6.689ng/ prl 36.893 ng/pl

Limit of qu:rntificxtiot ZA.27Ing/t n, t|.tgig/ltl

l're(:ision (t/oCV)

lnlr -day{n rt b.tuO-t.qZt - O.L,q_L.l;6

Intcr<lay (n:3) 0.345-1.987 O.2oj l.ssa

Accuracy (%)

Intra-d,ry (n:3) >98

lntcr-day (n=3) >97

>100

>99

% Recovcry (n:3)

Rohustncss Robu.st Robust

SpecificSpccificily Specilic

49

Tah le9

System Robustness Parameters

Prednisolon€ Acetate

Parameter o/. R.s-D Status

Flow Rate

Column Temperafure

Wavelength

0.218

0.436

0.175

Robust

Robust

Robust

Chloramphenicol

Flow Rate

Colurnn Temperatue

Wavelength

0.482

0.3'7 4

0.512

Robust

Robust

Robust

Table l0Recovery Studies of Preddsolone Acetate pharmaceutical Samples

Drug ilean 7o Recoverv % RSD

Pred +

25

50

75

t00.7

100.1

99.86

0.6t2

0.814

0.551

Prens

25

50

'75

93.2

97.9

99.1

0.013

0.128

0.461

Opthapred

25

50

101.0

100.8

100.3

0.532

0.134

0.612'75

Nlildopred

25

50

75

101.7

100.5

99.8

0.893

0.561

0.'197

Predforte

25

50

'75

97.8

100.6

102.5

0.'7 63

0.199

0.282

Table 1 i

Recovery Snrdies of Chloramphenicol Pharmaceutical Samples

Drug ilIean 1/o Recovery % RSD

Spersinicol

25

50

75

100.4

100.2

101.3

0.064

0.049

0.012

Chloroptic

25

50

75

98.6

99.1

100. i

0.087

0.123

0.516

Chloromvcetin

25

50

'75

99.9

99.6

98.9

0.349

0.043

0.025

Optachlor

25

50

'15

100.3

100.8

ta2.1,

0.27 |

0.061

0.058

Table L2

Recovery Studies of Phannaceuticxl Samples ( rontaidng Prednisolone Acetate andChloramphenicol in Combination

Drug NIeaD 9/o Recoverv % RSD

Predlrisvuth

25 99.8(Chlor)

96 2 (Pred)

0.162

0.501

50 97.6 (Chlor)

98.4 (Pred)

0.085

0.015

'75 100.6 (Ci or)

97.5 (Prel)

0.976

0.469

Predlricol

25 100.3 (Chlor)

99.3 (Pred)

0.5 81

0.482

50 100.9 (Chlor)

103.s (Pred)

0.260

0.018

'15 99.7 (Cblor)

95.4 (Pred)

1.0r2

0.649

Table 13

Alalysis ofPrednisolone Acetate alrd Chtoranr phenicol Pharmaceutical Products

Prednisolonc Acetate

Drug Dosage Fornr Nlean Con( ng/pl SD Yo Drug

Pred +

Pr€ns

Opthapred

Nlildopred

Predforte

Eye Drops

Eye Drops

Eye Drcps

Eye Drops

Eye Drops

853.00

865.01

g 15.87

987.48

953.61

0.5190

0.2178

0.3195

0.527'7

0.4563

85.5

86.5

91.6

98.7

95.3

Chloruphenicol

Drug Dosage Form iltean Conc ng/pl SD oh Dr,ug

Spersinicol

Chloroptic

Eye Drops

Eye Drops

918.333

9E6.2617

t02.2

79.086

t.5274

0.8765

0.4358

0.t236

91.8

98.6

t02.2

98.8

Chloromycetin Capsuie

Optachlor Ointment

Prednisolone Acetate and Chloramphenicol

Drug Dosage Form Nlean Conc ng/FL SD 7o Drug

Prednisynth Eye Drops 421.2 (chlor)

899.11 (pred)

358.272 (chlor)

970.7 (pred)

1.t60042

].9t694

0.24t6

1..160

105.3

89.9

89.5

9',7.1

Prednicol Eye Drops

2,1.1.2 Dexamcthasonc ancl Chloramphenicol

CoDlbiration of Dexalnethasono and Chloramphenicot ts cxtrnsivcly uscal to trert it)fectioits

associated with eye- Dexamethasone, 9 o-fluoro _ l l0, 17 o ,2l_trihydroxy 16 o nrethyl

pregna- t ,4-d iene-3,2o-dione is a potent fluorinated immunosupprcssive ard anliinflamnratory

agent, possess glucocorticoid activity, plays impo{ant role in the treatment of allergies,

plu, ^. o11i. oid res|or.ir e dcrmaro-e" a r.

Chloranrphenicol, D-thrco C) - 2,2- Dichloro, N, I p- hydroxy o. _(hyijroxynethyl) - p_nitro

phenyl acetamido , is the first natllrally occur ng nitrogen cootaiug conpound lhat shows

antibiotic activity, topically used for treatmerrt ofconjunctival infections. This combination is

Ior olfiLiJl sirh I SP. np or I uronedll phJIrd.opeir.

Dcxamelhasone a.d Chioramphenicot were dctermined,in pharnlaceuticat prepararions by

siuple and validated UPLC rnethod. Samples for ten plurmaceutical products were made ill

simple manner. UPLC mcthod was developed on Zorbex Eclipse XDB Rp Cl8 column (50 x

4.6 mm LD. , 1.8 pm) wiih chromatographic method parameters; 3.0 minLltes run time. linear

gmdient mobile phase ofacetonitrile and water, 0.ll mL/min flow rate, I pl_ injection volLxne

and multiwavelength detecrion at 238.5 and 278nm.'lhe dcvcloped nlctho.l was vatidatod as

per ICH guidclines for inter day and intraday precision, accuracy, linearity, specificiiy,

robushress, LOQ and LOI). Lincar response rvas achieved within the collcentration rangc ol.

50 i000 Lrg/mL, showed corrclation cocfficient of0.9998 and 0.9997 for Dexamefl]asone and

chloranphenicol respectively. Method was successfully applied ou tcn pharmaceutical

preparations of Dexamethasone and Chlorampheoicol including four eye drops and a tablet

formulation of Dexalrcthasone; two cye drops and capsule of Chloramphenicol; and cye

drops containing I)exancthasonc and Chlolamphenicol ir combination arrd was found

roliable for routine labomrory anatysis.

55

Optimization of Ultra Fast IIPLC Vlethod

With a view to deveiop i'eliabl,: and rapid method, IIPLC procedue was optimized

consirlering the injection volume, mobile phase florv rate, colurnn tempemture aad

wavelength. Five rnobile phase pr,lcedures were lested in dift'erent gradieflt ratios including

acetonitrile: 0.5 mr\I scdium acetate, ircctonit.ile: water, acetonitrile: 0.1 % formic acid,

methanol: water and methanol: 0.5 $,\1 sodiurll acetate Mobile phase having composition of

acetonitrile and water was found best. The peak at Rlof 2.550 1 0.0060 was recognized as

Chloramphenicol whiie the peak at Rr2.802 t 0.0065 was recognized as Dexamethasone in

comparison to the individual chrcmatograms obtained (Figure 8). The optimal DAD

wavelength (7. ma,.<)of for both Chk)ramphenicol and Dexarnethasonewas selected after

running the chlomatogram at five different wavelengths (238.5, 2'78,250,260.254). k,jectiott

volume of lpL was selected after ai]plying 3pL and 6pL injections. Colurtul tempemture of

25'C and io'C was checked but was lbund that 25"C was given higher tetention time.

Figure 8

Chromatoeram of Standard Chloramphenicol and Dexamethasone in

=l..1

l

:ll

Nlethod Validation

Linearity

The standard calibration curves in concentration nnges of 50_l000pg/ml for both

Dexamethasone and Chloramphenicol were found linear with corelation coefficient of

0.9998 and 0.9997 respectiveiy. Least squaxe rcgression data of Lhe calibration curves Iiom

triplicate injections of seven sta[dard working solutions were used to evaluate linearitv

(Table i5).

Precision and Accuracy

Inter-day and intla day coefficients of variation were observed at four conceltration levels

(100,200,600, 1000pg/ml). The accuacy was above 98 % for inter-day ard 99 % for intra

day precision. The %RSD is found to be iess thal 2 o/o in all inter day and intraday results.

The repeatability of sample application and measurement of peak area were expressed in

tfins of oZ RSD and the results are summarized in Table 16.

Limit ol Detection ond Linrit of Quontilication

For Dexamethasone, LOD and LOQ were found to be14.397 ng/pl and 43.632 og4u]- wl)tle

for Chioramphenicol, LOD ard LOQ were found to be 37.013 ng/pL a 112.161 n!1tL.

Rob ast n e ss a n d Specifi city

Selected parameterc (flow rate, column tempemtwe, and wavelength) of proposed method

was varied to 1 5oZ variation at tbree levels. Repiicate injections (n = 3) of standard solution

at thee concentation levels were performed. Peak purity for both Dexarnethasone and

Chioramphenicol was anallzed by con-rparing the chromatograms of stardard and

phan-Ilaceutical products. Ieak apex. peak start and peak end were taken under consideration.

No other peak was eluting at rRr of Dexamethasone andchloramphenicol. validation

parameters are summarized in Table 17.

Recovery studies

Pre - anal)zed pharmaceutical products werc checked lbr recovery studies with stafldard,

added up to 25 %, 50 0/o and 75 % ofdetected concentration and results are compiled in Table

l8- 19.

Analysis of Pharmaceutical Products

L'ltafast HPLC method at multiple wavelenth for detemination of Dexamethasoleand

Chloramphenicol in ten pharmaceuticai prcducts is developed for the first time (Figure 9).

Two peaks at Rpf 2.550 10.0060 a.1d Rr2.802 :0.0065 were identified in chromatogrcm as

Chloramphenicol and Dexamethasone in comparison to standard chromatogmm. No excipient

interference was observed in Dexamethasone and Clrloramphenicol individual

pharmaceutical products. Ma.jor degraded products were generated at Rro1D.571 In

Dexarnethasone ophthalmic suspension drug contents were found to be 97.2 o/o in Binadex,

99j yo in Methadex, 96.8 % in Ophthadex, 98.4 % in Spersadex and 99.7 yo it

Dexamethasone tablet. For Chloramphenicol eye drops, drug content was found to be 98.6 %

in Spersinicol, 99.5 o/o tn Chloroptic and 102.0 % in Chloromycetin.In combined

phamaceutical products of Dexarnethasone andClrloramphenicol, drug contents were

rcspectively found to be 94.0 yo ar1d, 9'7.5 yo In Dexoptic-C or,d 93.6 yo and 99.1 o/o in

Fluorbioptil. Low RSDTo reflected the suitability ofthe developed ultmfast HPLC method for

routine analysis of Chioramphenicol and Dexamethasone in pharrnaceutical products. Results

are summarized in Table 20.

I

Figure 9

Chromatogramsof Pharmaceutical Products ContainineDexamethasoneand

Chloramphenicol in Combination alonswith Standaxds

rl i E

60

Table 14

Gradient Progmm of Utta Fast HPLC Method for Determination of Dexamethasone

and Chloramphenicol

S# -T.rm-"

io ,t i^rto Solr,"rt,l Solvent B Flow rate Gradiedt

(MitliQ Acetonitrile ml/min

water)

0.380 Linear

2

0.8

60

25

60

130

40

15

3.0

4

0.8 Linear

1.5 Linear

.+0

20

0.8 Linear

0.8 Linear

Table 15

Linear Regression Data of De,(rrnethasone and Clloramphenicolfor Ultra Fast HPLC

Method

& Linear r! Regression Sloptr Intercept LOD LOQ

Range Equation

pdmI-

n! p,L ng';tL

D€xamethasone

2lo2 + 50-1000 0-99987 Y=6.589ix 10i- 0.7895 6.5891x10' 14.397 43.632

0.0065 0.1t95

Chloramphenicol

u -sso r so-rooo b.qsqz Y= 3.708 x l0r 8.2812 3.708x 10i 37.013 t12.l6l0.0060 + 8 .2812

Table 16

Aaalysis for repeatability and lntennediate Precision of Dexamethasone and

Ct oramphenicol (n : 3 for i nterday and 6 for intraday)

Dexamethis ,ne Chloramphetricol

lDtradav Prr.ci!ion I[tradav Precision

Conc

tdFL

100

200

600

1000

0.238

0.425

0_968

0.175

100.1

101.0

101.8

99.9

Conc

\!vL

100

200

600

1000

RSD % Accuracy 7o RSD % Accuracy Yn

0.629 105.1

0.'749 t02.4

1. r 60 101.3

0.'7'70 100.2

Interdav Precision Interdav Precision

Conc

ngFL

RSD % Accuracy 7o RSD % Accuracy YoConc

ndpL

100

200

600

1000

0.546

0.823

0.i67

0;72t

98.9

99.9

102.1

101.0

100

200

600

1000

t.7 52

0.20'7

1.1t2

0.83,1

102.1

101.1

10t.0

99.5

Table 17

Summary ofValidation Para meters

Parameters Dexamethasone Chloramphenicol

Linearity range .;0-1000 ng/pl 50-1000 ng/pl

Correlationcoefficient 0.9998

Limit ofdetection 14.397 ogpl-

Limit of quantification 43.632 ngp,L

0.9997

37.013 ng/pl

l12.161ndy,L

Precisior (yoc\)

Intra-day (n-3) 0.175-0.968

Inter-day (n=3) 0.3(;7-0.823

0.629-1.160

0.207 -1.752

- Accuracy (%o)

lntra-iay (n=3) >99

Inter-dav (n=3) >98

>100

>99

o/o Recovery (n:3)

Robustness

Specificitv

Robust

Specific

Robust

Specific

Table 18

Recovery Studies of Dcxamethasolre Pharnraceutical Sarnples

Drug Mean %, llecovery %, IISD

Ilinadc{

25

50

75

97.8

99.)

96.9

o_171

0.0IJ9

0.421

Nlethader

25

50

75

100.0

98.2

974

0.652

0.494

0.672

Opthadcx

25

50

75

99.0

102.0

s99

0.76t

0.3 89

0.512

25

50

t5

98.7

98.6

99.1

0.23t

0.084

0. t95

25

50

15

100.5

I04.5

t02.6

0.186

0.562

0.285

65

Table 19

Recovery Studics of Phalmaceutical Sanples conlaining Dexancthasonc and

Chloramphenicol in Combination

Drug NIcu 7o Rccovery % ]ISD

Dcxoptic-C

94.8(Chlor)

98 3 (Dexa)

96.5 (Chlo0

99.4 (Dcxa)

95.3 (Chlor)

97.2 (Dexa)

0 231

0.421

.s0 0.52l

0 625

0.4r1

0.81,1

75

l:luortrioplal

25 103.6(Chlor)

99.3 (Dexa)

99.8 (Chlo,

98.1 (Dexa)

99.4 (Chlor)

96.7 (Dcxa)

0.12t

0.952

0.684

0.129

0.951

0.483

50

15

66

TaL ie 20

Analysis of Dexametlasone and Chloranphenicol pharmaceutical producrs

Dexamethasone

Drug Dosage Fcrm Nleau Conc ngl1tl- -D - --

,7o D.1lg

Speriadex Eye Drops 192.0

Ophthadex Eye Drops 48"1.0

Nlethadex Eye Drops 496.5

Bhadex Eye Drops 486

Dexamethasone Tablet 498.5

0.611 98.4

0.259 96.8

0.513 99.3

0.064 97.2

0.815 99.7

Chloramphcnicol

Drug Dosage Form Nlean Conc SD o/o Drug

Spersinicol Eye Drops 986.51

Chloroptic Eye Drops 991.81

0.4'72 98.6

0.251 99.5

Chloromycetin Capsule 1020.07 0.351 102.0

Dexamethasone a[d Chloramphenicol

Drug Dosage Form Mean Conc SD o/o Dtng

Dexoptic-C Eye Drops 195.082 (chlor) 0.134 97.5

470.16 (dexa) 0.648 q4 0

Fluorbioptal Eye Drops 198.33 (chlor) 0.438 99.2

,168.35 (dexa) 0.261 93.'7

2.1.1.3 Dcxamethasonc, I'rednisolone acctatc and Chloramphcnicol

Dcxamethasone, Prednisolone Acctalc al1d Chlorarnphenicol \\,erc dctorlnined

sinlullaDeously fbr lhe llrsl lirre in seventeen pharmacoutical prodlrcts by llltra fast IIPLC

nrcthod. l-he proposed Ult.a Fast HPLC mcihod was developed on Zorbex Eciipse XDB Ri']

C18 colu n (50 x 4.6 rnm I-D. , 1.8 pn, in total run time of4.0 minutes Lrsing liroar gradicnl

nobile phase courposed o[aoetouitrile and waler, at flow rate of0.8 ml-/min, 1 ]rL injection

vollrme aod ,rlrlti photodiode affay dctcction at 254 nm. Following the recommended ICH

guidelines lor validation of devclopcd nethod, lincarity, accuraoy; inler day and intraday

prccis;on, specificity, robustress, LOD and LC)Q *cic validdtei:1. Ihe respon<e was linear

over the ooroertration lange of50-1000 pg/m1,, and correlation coefflcicnt of0.9999, 0.9999

ald 0.9997 for Dexenethasone, Prednisolone Acetate and Chloranrphenicol respectively

'vere obtained. Method was lound reliable for QC laboratory analysis and slrccessfully

applied on sevenleen pharmaceulical products inoluding Dexafiethasone eye drops and

tablet; Prednisolone acetate eye drops; Chloranlphenicol eye drops and capsUlc; and cyc

drops containing Dexamelhasone and Chlorarrphcnicol, and Prcdnisolonc Acetate and

Chloranrphenicol ir combination-

Optimization of Ultra Fast IIPLC Mcthod

ln order to devclop rcliable and rapid mcthod, UPLC proccdure was optimizecl considering

rl,c rrubilc plrdsc flo$ rJlc. ;,rte.rrorr rulurrr<..ulLrrrrr. I(r,f,irdrur. Jr,.r $d\rlcngrh. \rr.oL,..

nmbile phasc compositions were tcsted in differcnt gradient ratios using \.vater, methanol, 0-5

rnM sodium acetate, acetonitr;le, and 0.1 % formic acid. Firal seleclion was do[e for tho

mobile phase having composition ofacetonitrile and water. The peak at.(r of L496:l 0.0042

was recognized as Chlorarnphenicol. pcak at -R, of 2.186 I 0.00l5was recos,rizcd as

D€xarnethasone while pcak at 1ir of 2.685 1 0.0024 rccognizcd as I,rednisolonc Acclatc in

comparison to the indiliduais ckorlatogram obtained (Figue 10).254nm was chosen for

final selection after runr;ing the cluomatogram at five different wavelengths (243,

218,250,238.5,254). Injection volume c,f lpl- was sclected after appllng 3pL and 6pL

injections. ColuIDn temperatues of25oC and 30'C were checked but was found that 25'C has

given higher retentior1 time.

69

Figure 10

lo

Chromatoeram of Standard Dexamethasone. Prednisolone Acetate and

Chloramohenicol in Combination

Method Validation

Lineari6,

The standard calibntion curves in concentration ranges of 50-1000pg/ml for

Dexamethasone, Piednisoione Acetate and Chloramphenicoi were found linear with

corelation coefftci rt of 0.9991), 0.9999 and 0.9997 respectively. Least square reg-ession

data of the calibration cunes Ilom t.ipiicate injections of seven standard working solutions

were used to evaluate linearity, Table 22.

Precision and Accuracy

Inter-day and intra day coefiicients of variation were observed at four concenhation levels

(100, 200, 600, 1000pg/ml). the accuracy was above 977ofor both intra day and inter day

precision. The %RSD is found to be less t\an 2 %o in ail inter day and intraday results. The

repeatability ofsample application and measuement ofpeak arca were expressed in terms of

% RSD and the results are summarized in Table 23.

Limit of Detection and Limit of Quantirtcadon

For Dexamethasone, LOD and LOQ in ng/pl were found to be 1;1.684 and 44.195 while for

Prednisolone Acetate these were 6.689 a]l,d 20.217 rcspectively. For Chloramphenicol, LOD

and LOQ were 37.763 and 111.432.

Ro b,,stn ess afl d Sp e c ilicity

Selected parameters (flow rate, column temperatue, and wavelength) of proposed method

was varied to :! 5yo variation at dree levels. Replicate injections (n : 3) of standard solutiofl

at three concentration levels were perlbrmed. Peak purity for Dexamethasone, Prednisolone

Acetate andchio.amphenicol was anallzed by comparing the chromatograms of standard and

pharmaceutical products. Peak apex, peak start and peak end were taken under consideaation.

71

No other peak was eluting irt Rr of Dexamethasone, Prednisolone Acetate and

Clloramphenicol. Results ofvalidr fiion paramete$ arc summadzed in Table 24.

Recoverv studies

Pre - anallzed phannaceutical pr,rducts were checked for recovery studies with standard,

added up to 25 %, 50 yo aI,d'ii %, of delected concentration and oZ recovery was found above

94%.

Analysis of Pharmaceutical I'roducts

Ultafast 1IPLC method for detemination of Dexamethasone,Prednisolo[e Acetate ard

Chloramphenicol in seventcen phannaceutical products is developed for the filst time. Three

peaks at Rr of 1.496 _0.00.{2 was recognized as Chloramphenicol, peak at,Rr of 2.186

10.0016 was recognized as dexam€thasone while peak at R, of2.685:!0.0024 was recognized

as Prednisolone Acetatein comparison to standard ckouutogram. No excipient interference

was observed in Dexamethasone, Prednisolone Acetate andchloramphenicol individuai

pharmaceutical products. L1 Dexamethasone ophthalmic suspension drug contents were

found to be 98.1 % ir Binadex, 98.8 % in Melhadex, 91.1 yo in Ophthadex, 96.6 % in

Spersadex and 98.2 o in Dexamethasone tablet. ln Prednisolone Acetate ophthalmic

suspension drug contents were found to be 89.8 7o in Pred +, 91.2 9/o in Prens, 89.6 % in

Ophthapred,97.9 % ir Mildopred and 95.8 0% in Predforte. For Chloramphenicol eye drops,

drug content was found to 93.9 % ifl Spersinicoi, 99.\ yo tn Chloroptic and 99.4 yo tr

Chloromycetin. In combined pharmaceutical products of Dexarnethasone and

Cbloramphenicol, and Prednisolone Acetate and Chloramphenicoidrug contents were found

to be above 9.1 %. Low RSDo% reflecred the suitability of the developed uitmfast HPLC

method tbr sifrultaneous routine analysis of Dexamethasone, Prednisolone Acetate and

Chloramphenicol in phamaceuticai products. Results are surrmarized in Table 25-

72

Table 21

Gradient Programof Ulta Fa: t HPLC Method for Determination of Dexamethasone,

Prednisolone Acetate and Cltl rramphenicol

S.# Time irr miuutes Solvent A Solvent B Flow rate Cradient

( liltiQ Acetonitrile mI-/min

water)

I 0 8C 20 0.8 Linear

2 | 55 35 0.8 Linear

3 2 50 50 0.8 Linear

4 3 65 35 0.8 Linear

5 4 80 20 0.8 Linear

73

Table 22

Linear Regression Data of Dexamethasone, Predaisolone Acetate andCbloramphenicolfor Ultra Fast HPLC Niethod

R, ti""u. 12 Rcgression Slope Intercept LOD LOQ

Range

pYn[iiquation agl pL DelpL

Dexamethasone

2.186 50-1000 0.9999 Y=6.448x 10'- 0.7968 6.4'18x10-' t4.684 44.195

t0.001

6

J.7968

Prednisolone Acetate

2.685 50-1000 0.9999 Y= 7.387 x 10-1 1.7489 7.387 x 10-1 6.689 20.2?1

10.002 + 17489

4

Chloramphenicol

1.496- s0-1000 0.9997 Y= 3.708 x t0 8.362 3.708x 10' 3'1.763 111.432

0.0042+ 8.362

14

Table 23

Analysis for Repeatabilit y and lntermediate Precision of Dexamethasone.

Prednisolone Acetate and Ch orumphenicol (n : 3 for interday and 6 for intraday)

Dexamethasone Prednisolone Acetate Chloramphenicol

Intradav Precision Intradav Precision IntradaY Precision

Conc RSD 7o Accrrracy

g/FL ",6

Conc RSD Accuracy Conc RSD Accuracy

ng pL Y. o ngFL o Y.

100

200

600

1000

0.673

0.268

0.460

0.153

100.i

99.1

101.9

98.9

100

200

60u

1000

0.201

0.418

0.985

i 01.0

99.9

100.4

0.681 100.0

100 0.495 102.0

200 1.412 992

600 0.998 98.1

1000 0.639 100.0

IIlterduv Precision Interdav Precision Interday Precision

Conc RSD %o Accuracy Conc RSD Accuracy Conc RSD Accuracy

nC/pL oh orgpL

i00

200

600

1000

0.389

t.t42

0.281

0.689

100.0

100.1

98.1

101.1

okngtl, oh

100 1.648 101.1

200 0.876 98.1

600 t.32t 100.4

1000 0.951 97.0

r 00 0 .4t2 99 .2

200 0.641 100.0

600 0.864 98.5

1000 1.012 99.9

75

Table 24

Suurmary of Validation Paraile, ers

Parameters Dexi,trlethasone Prednisolone Acetate Chloramphenicol

Linearity rlnge 50-iC00 rglpl 50-1000 ng/pl 50-1000 ng/pl

Correlation 0.9999 0.9999 0.9991

coefficient

Limit of detectiotr 14.684 n:, pL 6.669ng/ pL 37.163 nd p'L

Limitof quantilication 44.19s ng/ pL 20.271n91 y'L 111.432nglpL

Precision (YoC\)

tntra-day (n=3) 0.153-0.673 0.201-0.985 0.495'1.412

Inter-dry (n=3) 0.281-1.142 0.412-1.012 0.876-1.648

-A.ccuracy (7o)

Intra-day (n=3) >98

Irter-day (n=3) >98

>99

>98

>98

>97

o/o Recovery (n:3)

Robusbress Robust Robust

Specificity Specilic Specific

Robust

Specific

l6

Analysis of Dexamethasonc,Pharnlaceutical Ploducts

Table 25

Pre.lnisolone Ao€tate and Chloramphenicol

1)rug Dosage tr'orn N{een CoIc st) % I)rug

Dcrlllncth.sonr and (lhloi-amphenirol

Dcxoptic-C Eye Drops

Irluorbioptrl Dyc Drops

188.01 (chlor)

4E0.04 (dexa)

19a.33 (chlor)

494.57 (dexa)

0.146

0.281

0.438

0.261

940

96.0

9',7.2

98.9

Prcdnisolone -A.cctate and Chlorampl'cnicol

l)rrg

Prednisynth

Prednicol Dye Drops

Dosagc Form Mean Conc

Eye Drops ,195.09 (chlor)

950.l5 (pred)

470.01 (chlor)

960.s6 (pred)

sl)

1.1785

1 .423

0.981

1.0120

'1, Drl.s

99.0

95.0

94.0

96.1

ll

2.1.2 Valiciated TLC - Densitometry Method for Determination of

Corticosteroid and Antibiotic in Pharmaceutical Products

7a

2.1.2.1 Prcdnisolone Acetato and Chloramphcnicol

,^.naccuratcandrelinbleHPTI-Cdensilometricmethodrvasdevelopedlotdeter]n;natiotlof

Prcdnisolole Acelate and Chloranpheoicol jn pharmaceulical preparations- Total ten

pharmaceutical products including eight individual products of Prednisolone Acetate and

Chloramphenicol and two ophthalmic susponsion containirg I'redrisolone Acctat€ and

Chloramphenicol in con'lbination were analyzed. Samples were spotted on IIPTLC silica gel

platcs60F254.Platewasdcvelopedusingchloroform:methanol(14:lv/v)andspotsat-Rr

0.21 1 0.02 and lli 0.41 1 0.03 rvere recognized as Chloramphenicol and Prednisolone

Acctate, respectively. !'or quantitative analysis densitometric measurenents ol thcse spols

were done at multiwavelength of 243nm al1d 278nm fhe dcvcloped lnelhod was validalcd

following ICH Guidelines Q1AR2 (Stability testing of new drug substances and products).

As the proposed method is accuralc and seleotive in effcctively separatiDg both the

coniponcnts, it may be applied in routine analysis ofthese pharmaceutical products.

Optimization of TLC Systcm

In order to develop a reliable stability indicating ILC densitometry melhod, solvcnt systenl

was optimized with standalds and pharmaccutical products. Different solvcnt systoms o[

chloroforln, methaooi, acetone, €ther, tolucDc,2-propanol and glacial acetic acid were

checkcd in varying ratios. Solve t systenls and rcsulting ?11 values of both standards are

sumrnarizcd in Table 26. Most ofthe solvent systcnls sho\a'ed diffused spots ofPrednisolone

Acetate. Suitablc scparation with bcst rcsolution Nas achieved Nith chloroform : mclhanol

(la : I v/v) q4rich showcd sharp bands with /?r value of Clhloranrpbcnicol a1 0.21 rL 0.02 and

ofPrednisolonc Acetato at 0.41 10.03 (Figurc 11).

Figure liChromatosram of Stardard (a) Prednisolone Acetate. (b) Chloramphenicoland

(c) Prednisolone Acetate and Chloramphenicol in Combination

Mcthod Validation

LineaiE

Standard calibration cuffe for bolh Prednisolonc Acetate and Chloranphcnicol in thc

concentration range of 200-6000 ng/spot was lound linear with I I S.D. 0.9966 ! 0.035 and

0.9920 ! 0.025, respcctively. I-;ncar regression data is compiled in Table 27.

ltrccisio and Accurac!

Intra-day precision lor prednisolone acetate and Chloramplrcnicol were Iound ro be Li2 and

l-09 while intcr-day prccision was found to be 3.90 and 1.68, rospcctivcly in terms of%

R,S,I),

I-imil of Detectiott and Lim of QuontiJicalion

For Prednisolone Acetate LOD and LOQ were Iound to be 0.0477 ng/spot aod 0.1446 ne/spot

whillr for Chloramphenicol thosc lvcre lound to bc aod 0.0300 n8/sPot and 0.0912 ng/spot,

respectively. Results ofvalidation parameters arc summarized in Tablo 28.

Robusltless a d SpeciJi.:il!

For robustncss analysis, the S.D. ofpcak area ofstandard lcvcls (200,400 and 1300 ng) was

estimaied fo. each parameter. For Prcdnisolone Aceiate and Chloramphenicol, S.D was 1.07

and 3.90 1br changing the amount ofn]obile phase, 2.48 arld 2.70 for varying in nrobile phase

composition, 0.95 and 2.96 for varying time from spotting to chromatography, 0.56 and 1 09

for vary,ng time frorn chroDatography to scanning and 1.53 and 1.32 for varynrg chambcr

satLrration time, respectively. SD for peak arcas were calculatcd and sum:narized in Table 28.

Peak puriry was estirrated by co,nparing the peak positions of both Prednisolonc Acetate and

Chloranphenicol in standard spectra wilh those in reaclion solutions. Good corfclation,

81

,"'z(staft, rniddle) = 0.999 and r,tniddle, end) = 0.9999 was obsewecl by conparing rhc

spcctra ofstandard and sa,nples, in both cases (Tablc 29).

Recovery Studies

Recovery studies sho.rved r.eoovcry of prednisotone Acetate (98_104%) and Chtoramphcnicot

(93-101%) fiom their phannaceutical products (Table 30).

Analysis of Markctcd Samplcs

Pharmaceutical producrs containinS both tlle active componrnnts i[ their individual and

combined pharmaceutical products were analyzed (Figurc 12 and l3). ln prcdnisolonc

Acetate eyc drops, Piednisolone Aoetate was found 97% (Ophrha It.ed), 100% (Mitdo hed),

9l% (Prcdfo(e),93% (Pred+) and 8l% (Prens) ofthe label claim. ln Chloramphenicol eyc

drops a[d capsule. lhc %s of Chloramphcnicot were found 98 (spcrsin;col), 95 (Chtoroptic)

and 99 (Chlormycetin). In eye drops, containilg prednisolone Aoetate and Clrloramphenicol

in combinatiorr, the %s ofChlorarnphenicol wcr€ 95 (Prednisynth) and 95 (prednicot) white

Prednisolone Acetate was iound to be 100% jn borh samples (Tablc 3l).

Figure 12

TLC Plate Showine the Spots of Standard Prednisolone Acetate.Chloramphenicol and Their lndividual and Combined Pharmaceutical Products

8l

rr ird6 &:r8 ln

Figure 13

Combined and Individual Pharmaceutical Products

Table 26

Optimization Data ofPredriso., xre Acetate and Cbloramphenicolfor TLCDensitometry Method

Solvent Systems (Ratio v/v) Rrof Xl ofPrednisolone Chloramphenicol

Acetate

2-Propanol : Toluene : Ether

(0.5: 1 : 8.5)

0.6010.02 0.43 10.02

Chloroform : Methanol : Glacial acetic acid 0.84 a 0.02 0.70 + 0.02

(8:1.4:0.6)

Toluene : Acetone

(6:4)

0.61 i 0.02 0.11 !0.02

2-Propanol : Chlorofqm : Glacial acetic acid 0.61 + 0.02 0.35 + 0.03

(1 : 9 : 0.05)

Cblorctbm : Acetone

(8:2)

Chloroform : Methanol

(14 : 1)

0.5410.02 0.21 10.03

0.41 i 0.03 0.21 t 0.02

8S

Table 27

Linear Regression Data ofpreCnisolone Acetate aod Chloramphenicol for TLCDensitometry N,lethod

Parameters Prednisolone Acetate

(at,t-u 2,l3nm)

Chloramphenicol

(a|,1*, 278 nm)

Linearity Ranp e

Correlation Cocflicicx t,

,21s1

Slope t SD

Intercept I SD

Y=rnx*c

200-6000 og/spot

0.9966 +0.035

2.42 !0.5

1106 - 0.48

2.42\ - 1lA6

200-6000 ng/spot

0.992010.025

2.74 !0.34

4183 12.1s

2.738x + 4183

86

Table 28

Summary of Validation Pari'rneters ofprednisolone Acetate and Chloramphenicol forTLC Densitomety Mrrthod

Parameters PrednisoloneAcetate Chloramphenicol

(at r.r. 243nn, (at1.",278 nm)

Linearity Range 200-6000 ne/spot 2OO+OOtXgspot

Correlation Coeflicient, 0.1t96ri 10.035 O.9g2O +0.025

r'1 sD

Y:mx+c 2.42x + 1106 2.738x+4183

ttrtra-day (n=3). % RSD l.l2

Irter-day (n=3), %o RSD 3.90

Limit of detection

Limit of qutrntilication

Specilicity

0.0477 ngspot

0.1446 ng/spot

Specific

1.09

r.68

0.030ongspot

0.0912 ngspot

Robust

Specific

Robustness Robust

Table 29

Summary of Robustresl j Pararneters

PrednisolotreAcetate Chtoramplinicol

Parameters

S.D. ofpeak area S.O-otpeat area

Amount ofNlobile Phase 1.0'7 3.90

2.70

2.96

1.32

NIobiIe Phase Compositio[ 2.218

Time from Spotting to Chronatography 0.95

Time from Chromatography to Scanning 0.56

Chamber Saturation Time 1.53

1.09

Average of three concentrations (20C.400 and 800 ng)

88

Table i ' )

Recovery Studies

Product Prednisolole Acetate

Yo llecovery

Chloramphenicol

7n Recovery

257550 50

ophth Pred 102.35 r0.24

99.14 a0.21

104.210.55

Spersinicol 94.26 !0.51

98.921 100.5510.12 0.1I

Pred1lis\,11th 99.52

0 t998.22 _0.24

100.2410.5 8

93.58 i0.28

100.82 1 9'7.2s !0.59 0.12

89

Tatrle 31

Amiysis of Pharmaceutical products

Product Pr€dIrisolone Acetatc Chloramphenicol

Conc. (in ng) 7o Dcgradation Conc. (in ng) 9/o Degradation

Ophth Pred

Mildo Pred

2909.94

3000

2778.6

3.00

0

Predforte

Pred+

7.38

6.-i62303.05

24,10.5

3000

3000

Prens

Spelsinicol

Chioroptic

Chlormycetin -

Prcdnisynth

Prednicol

i 8.65

2956.5

2850.9

2984.4

1t58.72

1).44.44

1.45

4.9'7

,1.63

t.52

3.440

0

2.1.2.2 l)cxamcthasone and Chloramphcnicol

A simple and accuratc FIPTLC dcnsitonretric method was developed lor determination of

Dexamelhasonc aod Chloramphenicol in pharmaccutical preparations. Oul of ten

pharmaceutical prodlrcts, two werc ophthalmic suspension containing Dexamethasonc a[d

Chloranrphenicol in combinat;on whilc the rest were individual products of D€xamcthasonc

and Chloramphenicol. Samples rvele spottcd or IIPTLC silica gcl platcs 60 | 254 and werc

developed in chlorolorrn: acetonc (8:2). After developrnent, Dexamethasone was found at ??f

!alLcor 0.404 n.027 d.,J Clrloralrplrenrcol sa" lorrnJal u.sl4 0.02u

,Iror quantitative analysis dcnsitornetric mcasurenents of these spols wcrc done at

multiwavelength of238.5 nm and 278nm. Irollowing ICH Guidelines QlAR2, the developed

method was validated. As the proposed method is accurate and selective in cfaectively

separating both the components, it may be uscd fo, .outir" urolysis of pharlnaceuticai

products containing these active components.

Optimization of TLC Systcm

With the ailn to develop a reliable stability iudicating method, solvent system was optilllizcd

with standards, samples and degraded products. Differerlt solvcnl systems oI chlorotorrlr,

mothanol, toluene aad acetoue were tried in varying ratios. Solvent syslenls and rcsulting.llf

vallres ofboth standards are summarized in Table 32. Suitable separation with best resolution

was achieved with chloroforn: acetone (8:2) {hich showed sharp bards with -1if valuo of

Dexamethasone at 0.40410.027 and of Chloramphenicol at 0.514 1 0.026 (Figrre l4).

91

Nlethod Yalidation

LinearitJ,

Stardard calibration curve for both Dexamethasone and Chloramphenicol in the

concenbation range of 200-6000 ng/spot was found linear with r21 S.D. 0-990910.002aid

0.99461 0.0021respectiveiy (Table 33).

Precision and Accutaqt

For Intra-day and inter-day precision, % R.S.D. observed for Dexarnethasone was 4.62 and

3.16, respectively while lor Chloramphenicol, 2.87and3..16, rcspectively.

Limit of Detection and Limit of QuantiJication

For Dexarnethasone and Chloramphenicol. LODs

0.005 tngspot, respectively while LOQS we.e lound

respectively (Tabie 3,1).

Ro b ustrrc ss a nd Specifi c ity

were tbund to be 0.0049ng/spotadd

to be 0.0l49ngspotand 0.0153ng/spot,

For robustness analysis, the S.D. of peak area of stardard levels (200, 400 and 800 ng) was

estimated for each parameter. S.D. was 1.85 and 2.51 for changing lhe amount of mobil€

phase, 1.43 aod 2.98 for varying in mobile phase composition, 0.63 and 1.15 for varying time

Aom spotting to chromatogBphy, 0.12 and 1.81 for varying time Aom ckomatography to

scaruing and 2.01 ard 1.35 for varying chamber saturation time ior Dexamethasone and

Chloramphenicol, respectively. SD for peal( areas were calculated and summarized in Table

35. Peak pufity was estimated by comparing the peak positions of both Dexamethasone and

Chloramphenicol in standard spectm with those in reaction solutions. Good conelation.

r':(srart, mirldie) = 0.9998 aird /'(iniddle, erd) = 0.9999 was observed by comparing the

specfta ofstandard and samples, in both cases.

Recovery Studies

Spiking studies showerl recovery of Dexanethasone (92-100yo) and Chloramphenicol (92-

99%) Aom ttreir pharmacrutical products (Table 36).

Analysis of Marketed S:tmples

hr Dexamethasone eye drops, Dexamethasone was lbund 97% (Spersadex), 96%

(Ophthadex), 94% (Nlethadex), 98% (Binadex) and 1009/0 (Dexamethsone Tablet) ofthe label

claim. In Chloramphenicol eye drops and capsule, the 7os of Chloramphenicol were found 98

(Spelsinicol), 93 (Chlorcptic) and 94 (Chlormycetin). In eye drops, contairdng

Dexamethasone and Chioramphenicol in combination, the %s of Chloramphenicol were 94

(Dexoptic-C) and 93 (Fluorbioptal) while Dexarnethasone wasfound 98 and 96%o in both the

sampies. respectively (Table 37).

93

Figure 14

TLC Plate Showine Spots ofStandard Dexamethasone. Chloramohenicol andDexamethasone and Chloramphenicol in Combination alonq with Their

Pharmaceutical Products

931

Table 32

Optimization Data ofDexat:ethasone and Chioramphenicol for TLC DensitometryNlethod

Solvent Systems (Ratio v/v) n;?D;*u-ethu.o"" X,ofChloramphericol

Toluene : Acetone 0.26):0.02 0.3110.02

(10:5.05)

Chloroform : Acetore 0.27X0.02 0.33+ 0.03

(11:4)

Chloroform : NlethaIol 0.41 10.03 0.21 + 0.02

(14 : 1 2"d run )

95

Table 33

i-inear R,:gression Data of Dexa methasone andChloramphenicol for TLCI)eositc,rrletry Method

Par amer:ers Dexamethasone

(at ;,,,"*238.5 rm)

Chlorrmphedcol

(atl*,278 trm)

Linearity Range

Correlatio[ Coeffi cient,

"21sD

Slope i SD

Intercept j SD

Y:nlx+c

2()0-6000 ng/spot

0.9909-!0.002

1.33,1+ 0.0398

2717 .510.459

200-6000 ng/spot

0.994610.0021

1369+0.0297

3319+0.063

1.369x + 33191.334x t 2117

96

Summary of Validatiou Par ,uneters

Densitonetry N{ethod

Table34

of DexametlLasone and Chloramphenicol for TLC

Parameters Deramethasone

(at;*,238.5 nm)

Chloramphenicol

(at,lh., 278 nE)

Linearity Range 20C-6000 ng/spot 200-6000 ng/spot

Correlation Coeflicierlq

r,1SD

Y=IlLx+c

Intra-day (tr=3), 70 RSD

Inter-d:ry (rF3), 7o RSD

Limit of detectioD

Limit of quantification

RobustDess

Specincitv

0.9909+0.002

1.334x + 2'71'7

4.62

3.16

0.0049ngspot

0.01,+9ngspot

Robust

Spccific

0.9946+0.0021

1.369x + 3319

3.16

0.0051 ngspot

0.0153ng./spot

Robust

Specific

9t

Table 35

Summary ofRobusl ress Paramete6

Dexamethasone Chloramphenicol

Parameters

S.D. ofpeak area S.D. ofpeak area

Amount of Mobile Phase

Chamber Saturation Time

1.85 2.51

1.43 2.98

2.01 1.35

Mobile Phase Compositio[

Time from Spotting to Chromatography 0.61 I 15

Time from Chromatography to Sca[ning 0.12 1.81

Average ofthree coDcenrmtions (200.400 and 800 ng)

Table36

Recovery Studies

De\amethasolre

%o Recovery

Chlonmphenicol

7o Recovery

Spersadex

Spersinicol

Dexoptic-C

100.28i0.16

94.061

0.18

98.231

0.04

93.6610.68

97.52!o.2t

98.871

0.39

92.71L0.42

100.011

0.'7 4

96.93!0.37

92.14!0.'72

95.s31

0.63

99.481

0.61

Table3T

Analysis of Pharmaceutical Products

Product Dexamethasone Chloramphedicol

Couc. (in ng) 7o Degradation Conc. (in ng) 7o Degradation

Spersadex

Ophthadex

Methadex

Binadex

Dexamedrasone

Tablet

Spersinicoi

Chloroptic

Chlormyceti11

Dexoptic - C

Fluorbioptal

281',7.9

2951.2

118.29

2909.9

2893.4

3.00

3.55

6.07

t.52

0

1.41

3.63

1.16

2956.',]

2891.0

7.03

3.36

5.86

100

2965.0.\

2139.0

2899.4

564.8

I t25.2

2.1.2.3 Dcxamethasone, PrcdnisoloneAcctatc and Chloramphenicol

A simplc and accurale HPTLC densitornetric method wlrs developed lor determination of

Dcxanethasonc, Prcdnisolone Aoetate and Chloramphcnicol in pharmaccutical preparations.

Out of seventeen pharDraccutical prodlrcts, fo r wcr€ ophthalnric suspension conraining

Dcxamcthasonc and Chloramphcnicol, and Itednisolonc ,^cetate and Chlorarrphcnicol in

combinalion while the resl were individual prodLrcts ofDcxamcthasone, Prednisolone ,{cetate

and Clilorarrphenicol. Sanrples werc spotted on IIPTLC silica gel plales 60 I254 and were

dcvcloped in chlorolbrm: melhanol (14:l 2nd run). After dcvelopmenl, Dexamcthasonc was

found at Ir value of 0.18 1 0.02, Chloranrphenicol was found ar 0.2I | 0.01 aDd Prednisolons

Acctatc was found at 0.38 ! 0.02.

For quantitative analysis densitometric measurements of thcsc spots werc donc at

multiwavelength of 238.9nm,2,13 nm and 278 nm. Following ICH Cuidclincs QlAIt2. thc

devclopcd ncthod was validated. As lhe proposed mclhod is accumtc and selccli\e ill

cflcclivcly separating both thc coftponents, it may bc used lor roulino analysis ol

pharnaccLrtical products containing these active components.

Optimization of TLC System

With the aim lo del,clop a retiable stability indicathg method, solvent svstcn was optiruized

\vith stalldards, samples and degraded products. Dillcrcnt solvcnt systems of chlorotbrm,

methanol, 2-propanol, toluenc, glacial acetic acid, €ther and acetone were tried in var)in3

ratios. Suitablc separation with best rcsolution was achieved witb chlo.oform: methanol (14:l

2nd run) which showcd sharp bands with llr valuc of L)examcthasonc a1 0.18 | 0.02,

Chloramphenicol a1 0.21 10.0i and olPrednisoloneAcetateal 0.i8f 0.02.

101

Method Validalion

LincdriU

Standard calibr:ltion curve lor Dexamerhasore, prednisolone Acetate and Chloramphenicol in

the concentration range 0f200-6000 ng/spot was lound linear with I t S.D. 0.9966 I 0.035,

4.9920 ! 0.A26 and 0.9981 1 0.024 respecrively (Tabte 3 8).

Precbiot, and Acculacj

Iror Int|a day and intcFday preoision,,% R.S.D. obscrverl

2.91, lor l,rednisolone Acdiale 2.38 and 1.04 while fort

rcspectivcly.

lor Dcxamcihasonc was i.87 and

Chloranrpheniool, 2.19 and 2.08,

Lit til of Deteclion and Litltit of eudhtifrcation

lor Dcxanlethasonc, Prednisolone Acetatc and Chloramphenicol, l_ODs wcre lound to bc

0.088 ng/spot, 0.0is ng/spot and 0.029 nglspor, respectivety while LOes were found ro be

0.269 ng/spot, 0.107 ng/spot and 0.088 ng/spot, respectively. Rcsults ol validation paramclers

arc sunrmarized in Table 39.

R0hustness ul SpeLilicilJl

for robustness analysis, the S.D. ofpeak area ofstandard leveis (200, 400 and g00 ng) was

estinlatcd for each paramcter. S.D. was 1.03, 1.75 and 2.68 For changing the amounr of

nrobile phase, 1.99,2.42 and2.61 for varying in mobile phase conrposition, 1.62, 0.85 and

1.96 for varying time fronr spott;ng to chromatography, 0.49, 0.63 and 1.75 tor varyjng rimc

liom chlomatography to scant ng and 2.11, l.1i and 2.01 for varying chantbcr saturatio0

time for Dexanethasone. Prednisolone Acctate and Chloramphcnicol, rcspccrivoly. I,cak

purity was estiurated by comparing the p(dk positions ot r)cxanruthusonr. ltcdnisoronc

Acetatc and Chloranphenicol in standad spectra with thoso in reaction solutions_ Cood

742

coffelalion, r'?(start, middle) = 0.9996 alld l1,riaat., end) - 0.999g was obscrvcd by

corrpdrill- thc spe,lra ol.tJ dard anJ satnplcs. in bolh cdscs.

Recover-y Stadies

Spiking studies showed recovery of Dexamerhasone (92-tOt%), prednisotone Acetatc (95

101%) and Chloramphcnicol (94-98%) lrom their phamlaceulical producrs (-table 40).

Analysis oI Marketcd Sanlplcs

In Dexametlrasone pharmaceutical products, active conlponcnl was found morc than 90 o% of

fhe label claim. In Prednisolonc Acetate pharmaceutical products, Prednisolone Acctate was

found more than 94%ofthe label claim. In Chloramphen;col p.oducts, % of Chloramphen icot

was found above 95%. l[ eye drops, containing Dexamcthasone and Chloran]phcnicol in

combination and Prednisolone Acctate and Chlolarnphenicol in conrbination, lhc o/. purity ol

active componcnls wcro lound abovc 94% (Iable 41).

103

Table 38

Linear Regression Data of Dexamethasone, prednisolone AcetateandChloramphenicol for TLC Densitomery Method

Parameters Dexamethasone Predtrisolone Cntoramplenicot(a1r..",238.5 nm) Acetate (at,I.,, 278 nm)

(at;.,,243 nm)

Linearit-v Range 200-6000 ng/spot 200-6000 ng/spot 200-6000 ngspot

Correlation 0.996610.035 0.9920 j0.026 0.9981+0.024Coeflicient,

"21sD

Slope 1 SD 1.29910.0398 2.12 t0.45 2.'t2+ 0.35

Irtercept l SD 2711!).459 1108:_ 0.47 418512.08

Y=mx+c 1.299x -2711 2.,12x-1108 2.72x + 4185

Table39

Summary of Validation parameters of Dexamethasone, prednisolone AcetateandChloramphenicol for TLC Dersitomerry Method

Parameters Deramethasone freanisotone --- Cn[rampleaicot(at,l.*,238.5 nm) Acetate (at,l@r 278 nm)

(at,l*,243 nm)

Linearity Ratrge 200-6000 ngspot 200-6000 ng^p;a-- 100_60 nghpot

Correlatior 0.996610.035 0.992010.026 0.9981{.024Coeflicient,/"1SD

Y=mx+c 1.299x+21ll 2.42x+ 1108 2.72x+ 4185

Intra-day (tr=3), 7o 3.87RSD

2.-3 8 2.19

lnter-day (r=3), Yo 2.91RSD

3.04

Robusr

2.08

Robust

Limit of detection 0.088ngspor 0.035ngspot 0.029ng/spot

Limit of 0.269n9/spot 0.107ng/spot 0.0882n9/spotquantilicatioa

Robustness Robust

Specilicity Speciflc Specific Specific

Table40

Recovery Studies

Product DexamethasoneYo Recoyery

50

Predtrisolone Acetate7o Recovery

Chloramphenicol7o Recovery

94.991 98.42 98.250.75

Spersade 100.68 92.62! 97.08x + 0.09 0.34 t

0.17

Ophthapred

110.16 0.34

Dexoptic- 97.50+ 94.7210.c 0.14 92

Prednisynrh

97 .25!0. 94 .01 96.4824 1t

0.29 0.84

96.02! 97.26 95.080.89 1 1

0.16 0.49

98.20

I0.61

95.42 10t.47 91.53

: -! 0.,11 i0.91 0.28

9',1.41 97.28+ 98.23

:: 0.56 10.17 0.75

75 25

Spersinic -ol

Table4l

Aaalysis of Pharmacerrtical Products

Dexamethasone Prednisolone

AcetateChloramphenicol

'4 Degiadatiod 7o Dcgradation 9/o Degradation

Spersadex 4.01

Ophthadex 2.91

Methadex

Binadex

3.78

5.36

1.84

2.t9

5.24

3.51

1.24

1.75

2.22

2.10

1.91

t.45

4.97

0.52

5.61

2.94

Dexamethasone 4.19

Tablet

Prcd +

Prens

Ophthapred

Mildopred

Predforte

Spersinicol

Chlorcptic

Chlormycetin

Dexoptic - C

Fluorbioptal

Prednislmth

Prednicol

3.7 |

21.83

2.2 ICH RECOTVIIVIENDED STRESS DEGRADTION

STUDIES ANID DEVELOPMENT OF VALIDATED

STABILITY INDICATING TLC - DENSITOMETRY

METHODS FOR QUANTIFICATION OF

CORTICOSTEROID AND ANTIBIOTIC,

INDIVIDUALLY AND IN COMBINATION

108

2.2.1 Prednisolone Acetate, Chloramphenicol and Both in Combination

Literure survey revealed that simultaneous detsrmination of Predtisolone, Chloramphenicol

and a degraded product, 2- amino- 1-(4-nitrophenyl)propane- 1,3-dio1 in ophthalmic solutions

tkough HPLC is reporredt55l. Few ffiTLC methods arc aiso found reported for the

determination of Chloramphenicol in combination with various different compounds

separately like benzocaine and 2-amino-l-(4-nitrophenyl) propane- 1,3-diol (ANpD), and

with dil'ferent corticosteroids inciuding Prednisolone, Prednisolone Sodium

Phosphate,Dexamethasonesodium Phosphate, Betamethasone Sodium phosphate,

Hydrocortisone, Hydrocortisone Acetate and Betamethasone- 17-Valerate tel. Simultaneous

determination of Prednisolone Acetate and Chloramphenicol in the presence of their

degraded products though Tlc-densitometry has not been reported so far.

Validated, simple and accurate Tl-c-densitometry m"thod [u"66] for the simultaneous

deterrnination of Prednisolone Acetate and Chloramphenicol in presence of their degraded

products Oy applying stress corditions)was developed, following ICH guidelines Q1A in

consideratio[. As ail the pharmaceutical products are supposed to b€ assayed for potency, a

validated Tlc-densitometry method, that do not show any interferences of degraded products

with the drug active components is useful in measuring these components in routine analysis.

The developed TLC method can be used for routine analysis of Prednisolone Acetate and

Chloramphenicol in presence of their degraded products in their individual and combined

pharmaceutical formulatiolls.

Optimization of TLC System and Nlethod Validation

Solvent system was optimized with standards, samples and degraded products. Different

solvent systems ofacetone, methalol.2-propanol, chloroform,toluene, ether and glacial acetic

acid were tried in varying ratios. Most of the solvent systems showed diffused spots of

Pred,,.olone Acelare Suirrblc \epdr,ri"rr *irtrbc,rre,olu,io.w.r.J.hi.!(Juirt,.trtorotbrnr

: methanol(14 : I v/v) which showed sharp bands with llrvaluc oI Chloranphen icol at 0.2l

1' 0.02 and of Prednisolone Acctate at 0.41 I 0.0i3. Standard calibration clrrve of both

Prcdnisolonc Acetate and Chlommphenicol rvas lound lincar in the concentration rangc of

200 6000 ng/spot wirh ,'1 S.D. 0.9966 1 0.035 and 0.99201 0.025, rcspectivcly. For Intra_

day and iDter-day precisioD, % R.S.D. observed for prcdnisotone Acorate was i.l2 and 1.90,

rcspeotivr:ly while lor Chloramphenicol, 1.09 and 1.6g, respcctively. ltr I)rodnisolonc

Acctate and Chloranphcnicol, LOD were 0.0477 ng/spot and 0.0300 ng/spot rcspccrivcly

whilc I-OQ were 0.1446 ng/spor and 0.0912 ng/spol, respectivcly. Ireal( purity was estimared

by comparing the peak positions of both prednisolone Acetatc and Chloranrphcnicol in

standard spectra with thosc in reaction solutions. Cood eorrelation, 17 (stalt, mrddle) 0.9999

and /' (Iniddle, end) = 0.9999 was observed by comparing thc spectra of standard and

samplcs, in both cases- Thc linear rcgrcssion data and tho rnethod validation paraDctcrs are

sunrmarized in Table 42.

Stability Indicating l,ropcrty in Prednisolonc Acctatc

Acidic, alkaline and neutral hydrolysos condirions, wc1 hcat and phorochemicai conditions

showcd grealer dcgradation cffeots to Prednisolonc Acetate. Undcr various strcss condilions

wilh some comlnon and different pcaks, total eightoen degradeil produds wcrc obscrvcd

Peaks with lower,4f values indicated high polar nature than prednisolone Acetato.

lcidic llydrolysis

In lN IICI, Prednisolone Acetate was deconposed ry to '16.30/o whilc io 5N HCl, it

decomposcd upto 100%.'ihree additional peal(s ar /tf 0.01,0.0j and 029 wcrc conrnonty

gencrated in both acidic rcaction mixrures. In addition to thosc pcaks, onty iD IN IICt

reaotion mixture, thrcc additional peaks at /lr 0.17,0.i9 and 0.73 lver€ also gcncrared whilc

110

four additional peaks at 1ir 0.02, 0.58, 0.78 and 0.80 werc onl) obtained under strong acidic

condition (5N HCI).

Alkalihe H!drolJ,sis

100% degradation was observed under alkaline conditions. Degraded peaks were observed at

nf 0.0i,0.02 and 0.50. In addition to colnmon peaks! in 0.lN NaOH, three additional peaks

were also observed at Rr 0.17, 0.36 and 0.78, and two additional peaks at l?r 0.03 and 0.29 in

lN and 5N NaOH treated solutions, respectively.

Neutral Hydrolysis

Unde. neulral hydrolysis conditions, four additional peaks at 1?r 0.01, 0.02. 0.17 and 0.29

were generated and Prcdnisolone Acetate was degraded upto 77.loZ.

Wet ahd Dry llcal Degtadatiot

Prednisolone Acetate showed 95.go/. and 18.60/. degradation urder wet heat and dry heat

degradation conditions, respectively. Both cqnditions showed common degraded peaks at,r?r

0.01, 0.17, 0.26 and 0.47, while additional peaks we.e observed at .Ri 0.29, 0.46 and 0.80 for

wet heating and at Rr0.78 for dry healing condiaiorls.

Oxidation Reaction

One ofthe oxidation mixtures rvas refluxed for 2 hours ar 80'C tvhile lhe second was kepl ior

24 hours at rcom temperature and showed 21.19/o and 5.50/o degradation, respectively. Under

both the oxidation conditions, three common peaks were generated at nr0.05. 0.i,1. and 0 29

P h olo D e g r ad at io h C o nd ilio rr s

i00% dcgradation $'as observed under photochemical condiiions and degraded peaks werc

generated at ,Ar 0.0l, 0.29, 0.31. 0.47, 0-58 aDd 0.71.

111

Under all the strcss conditions excopt oxidation reaction, a common degraded product wjth 1ll

0.01 was generated. Dcgraded product with Rf 0.02 was only generated by acidic (5N HCI),

alkaline (0.1, I and 5N Naoll) and neutral hydrolysis. Peak at .4r 0.03 was gcneraled under

acidic (l and 5N HCI) and alkaline (lN NaOH) hydrolysis while peaks at 1lr 0.05 and 0.14

were only obtaired by oxidation reaction. Acidic (lN tICl), alkaline (0.i N NaOH) and

neutral hydrolysis and wet heat and dry heat degradation conditions commonly generated a

degraded product at nr 0.17, while peak at .4r 0.26 u,as only lound by wet and dry hearing

conditions. Peak atni0.29 was lound in various stress conditions iroluding acidic (l and 5N

HCI), alkaline (l and 5N NaOFI) and neutral hydrolysis, wet heating, oxidation and photo

degradation. Characteriit;c peaks ofphoto degradation and alkaline hydrolysis (0-l N NaOH)

were found at ,Rr 0.3J and 0.36, respcctively. Similarly peak at Rr 0.46 was only generated in

wet heat degradation rcaclion, while a peal( with ,4r 0.47 was obscNed in wet and dry heating

and photo degradation conditions. Dcgradcd product at 1af 0.58 was generated by acidic

hydrolysis (5N HCI) and photo degradation. Pcak with l?r 0.73 was found in acidic hydrolysis

(iN HCI) and photo degradation. Acidic hydrolysis (5N HCI), alkaline hydrolysis (0.1N

NaOll) and dry hcating commonly generated a degraded peak at,R6 0.78. Dograded product

at.Rr 0.80 was senerated through ac;dic hydrolysis (5N HCI) and wet heal dcgradation. Video

densitogram pictures are shown in Figuie 15 while chrotnatograms of stress dcgraded

products obtained from Prednisolone Acetate are shown in Figures l6 21.

Slability Indicaling Property in Chloramphenicol

Chloramplrenicol showed greater susccptibil;ty to degradatior to alkaline, $rct hcating and

photochemioal stress conditions.

71?

Acidic Hydrolysk

In 1N HCI and 5N HCl,ClLloramphenicol was degaded upto23.8oz and 69.4 yo. Corffnon

degraded peaks were observed at Rr r).01 and 0.04, while in stong acidic medium (5N

HCI)an additional peak was E;enerated atni0.31.

Alkaline Hydrolysis

Chloramphenicol was 100 % ciegraded under ail alkalioe conditions and a common degraded

peak at ,Rr 0.01 rvas generated. Under 0.1 N NaOH alkaline conditions, additional degraded

products werc observed at Rr 0.4-1 and 0.79, while peaks at RP.04 and 0.81 were also found

in lN NaOH fteated solutior. Tluee additional peaks at ,Rf 0.02, 0.04 and 0.48 were found in

5N NaOH treated soiution.

Neutral Hldrollsis

Under neutral hydrolysis a single degraded product was found at Rf0.01.

Wet Heat and Dry Heat Degradation

Two cornmon additional peaks were found in wet and dry heat degadation conditions at Rr

0.01 and 0.79.

Oxidstion Reuctions

Under both the oxidation conditions, three additional peaks at R1 0.01, 0.12 and 0.31 were

generated.

Ph otoc h e n ic al Reac tio n

Under photochemicai degradation conditions, seven additional peaks at R10.01, 0.09, 0.15,

0.27, 0.44, 0.81 and 0.85 were geDerated.

Under all stressed conditions, degraded product with ?l;0-01 was corrmonly fbrrred while

de8raded peak with lir 0.02 was only observed under alkaline hydrolysis (5N NaOII). Peak

with ,Rr 0.04 was gcneratcd by acidic (l and 5N HCI) arrd alkalinc hydrolysis (l and 5N

NaOH), while pcak at ??f 0.48 was observed only in alkaline hydrolysis (5N NaOH).

Degraded products with ,4.0.12 and 0.15.were found under oxidation and photo degradation

conditions, respcctivcly, while peak with .Rr 0.31 was formed only in oxidation reaction.

Undcr all(alinc hydrolysis (0.1N NaOll) and photo de8radation reactions, peak with 1il 0.44

was gcncratcd. Degraded pcak with Rr 0.79 was generated by alkaline hydrolysis (0.1N

NaOH) and wet and dry heat deSradations. Under acidic hydrolysis (5N HCI), alkaline

hydrolysis (lN NaOH) and photo degradation conditions, dcgradcd product with 1lf0.8l was

generated. Similarly peaks with'Rf0.09, 0.27 and 0.85 were only generated throush photo

,{de8radation reaction. Video densitogram pictures are shown in Figure l5 and chrofiatograms

ofChloramphcrricol strcss dcgladef,products are shown in Figures 16-2l.

Stabilily Iudicaling Property

Chloramphcnicol in Combination

Acidic llydrolysis

Prcdnisolone Acetatc was degraded upto 100 % under both acidic condilions. In conrparison

with the individual standard solutions, a combined standard solutions showed two add;tional

peaks under acidic (lN HCI) condition at nf 0.78 and 0.61 while three degmded products of

Prednisolonc Acetate at & 0.03, 0.19 and 0.73 were rnissing. Peak at -Rf 0.81 corresponds to

the degraded product of Chloramphenicol. Two additional peaks at ni 0.17 and 0.38 were

found undcr 5N HCI stress condition while degradcd products of Prednisolone Acetate with

nr 0.01 and 0.80 were missi,rg.

Prednisolonc Acclrlc and

114

Alkaline Hydrolysis

Chloramphenicol aad Prednisolone Acetate were completely degraded under alkaline

conditions. Two common additional peaks were generaterl under all alkaline cotditions at nr

0.01 aad 0.02. Under 0.1N NaOH stress condition, two additional peaks were found at Ri

0.29 and 0.85 while Prednisolone Acetate degraded peaks at R1 0.36 and O.7g ard

Chlommphenicol degraded peak at Ri 0.44 were missing. Moreover, under lN NaOH stress

condition, afl additiotal peak was obse ed at RO.44 wfule Chloramphenicol degraded

prcducts at Ri 0.04 and 0.81 and Prednisolone Acetate degaded product at Rr0.5O were

missing. In 5N NaOH treated solution. comp.lrison showed an additional peak at Xr 0.31

while Chloramphenicol degraded products at Rf 0.04 and 0.48 and prcdnisolone Acetate

degraded products:Lr R, 0.29 and 0.50 were missrng.

Neutral Hydtolysis

In neutlal hydrolysis reaction mixture, peak for Prednisolone Acetate at Rr 0.29 was missing

and an additional peak was genemted at Rf0.79.

Wet and Dry Hest Degradation

An additional degaded products at Rr 0.50 was observed in combined sample urder \ ret heat

degradation while degraded products of Prednisolone Acetate at R&.26, 0.46,0.47 and 0.80

were missing. Under dry heat degadation condition, comparison showed an additional peak

at -Rr 0.73 while Prednisolone Acetate deg€ded peaks at R6 0.17 and 0.78 were missing.

Under wet heat degadation condition, Prednisolone Acetate was 177o degraded while

Chloramphenicol was degraded up to 36.9 %.

Oxid.ation Reaction

For oxidation reactiofl, two conditions were applied.Under oxidation condition (refluxe<l for

two hours at 80 oC), an additional peak at ,Rr 0.55 was observed while two additional peaks at

Rf0.23 and 0.55 were obsa'ved under second oxidation condition (reaction mixture kept for

24 hous at rcom temperature). Prednisolone Acetate degraded peaks at Ri 0.05, O.l4 and

0.29 were missing.

Photochemical Degradation Co nditions

Under photochemicai degradarion condition, additional degmded products at _RO.49.0.50 ard

0.69 were generated only in combined sample whiie a degaded products of prednisolone

Acetate at Rr 0.29 and 0.33 ard those of Chloramphenicol at rtr 0.09, 0.15, 0.28, 0.44, 0.g1

and 0.85 were missing.

Stress degradation study of Prednisolo[e Acetate and Chloramphenicol in individual and

combined sampies is summarizerl in Table 43 and 44while video densitog.am pictuIes are

showl in Figure 15.

Stress degmdatio[ study data showed that under all stress co[ditions,peak with R|0.0i was

commonly tbrmed. Degraded products with Rf 0.09 and 0.15 were formed by

photodegrdation. Degraded products wifi Rf 0.38 and 0.58 were not found under any stress

condition applied to Chloramphenicol alone. Degraded product with Rr 0.38 was generated ir

combined sample of Prednisolone Acetate and Chioramphenicol under 5N HClacidic

hydrolysis (Figures l6-21)

Analvsis of Nlarketed Samples

Two expired pharmaceurical products were also analyzed. Figue 22. In expired sample

(spersinicol), peak was not fourd for Chloramphenicol at Rf 0.21 (100 7" degradation) but six

degraded peaks were found at ,It10.01, 0.09, 0.15, 0.19, 0.38 aIId 0.5g.In expired prednisynth

prcduct (light yellow in co'or), peak for Chloramphenicol was found but with 59 %

degradation and Prcd1isolone. Acetate was d egiad,ed, lp to 12.2 yo. Five adrJitional peaks were

also found at Rr 0.01, 0.I J, C.26, 0.38 and 0.58. Compa.isort showed that degaded product at

R6 0.01 was a common pro,:uc t in bot[ individual and in combination. Deg.aded products at

rR10.15 and 0.38 were generarred by the degadation of Chlorumphenicol. Degaded Foducts

rvith R6 0.26 (lbund under dJ and wet heat degradation conditions) ard 0.58 (produced under

acidic hydrolysis and photo degadation) were associated with the degadation of

Predlisolone,A.cetate.

I

r. la. , tl tt. t{ ti. ta, rr, ta

I

!.{ I r. la. rl. 11 rl ra. lt. ra. tt. tr

Figure 15

TLC Plate Showine Soots of Standaxd Chloramohenicol. Prednisolone Acetate.

Chloramphenicol and Prednisolone Acetate in Combination (A)lN HC l. 5N HCl.0.lN NaOH. lN NaOH. 5N NaOH (B) Neutal Hvdrotysis. Wet Heatine. Dry

Heatine. Photo Desradation and Oxidation at Room Temperature

l1ts

a- iD .-

(Bl

EE

=

,r.l

oc

o

o1

oJaal0orc

a

o-

ac

=

N

N-a

-a

I

N)\]

=c-

--

aa

a-

-

?-'a

l.J-]

=v.

2a!.

a-

C.

a-c?.

-

IFJlrol-lo l:/

OQl-mth tlDaD l=

t-- L-.

arll ldlc l=rD lq+- lO

lAot-. l,la lao

It' 13-t(D!!-

l{-too I Fn

lg=lx a

l^r={l= L-

lY. LNJ

ln la

l+ tElo=I? LEl(} li.lc- lir

l- ho

L>'

aD lNrgt;

=Z

El

aloa. t=

lc 0a

/l=

1dR

l-l x Oc

= l.E. t\)

= l'!

{,1^lc ll.J1=^aj' L

=li

t:. =olxo- lo)

to-l,/ =lEx

lq lN

rl-c- l=

I

l

I

=T

.ri&'-

\c

Ca

I'J

=l.)

-a

3cf

=a

I

IJ-l

-

c-

4

;'.-?d

cF6-

C-

-

:,:

tJ

=a,l.L;

i=

ala.J-

ra.i=;+a

lc.l5

9l=ls

=

t3 la

!4 loo0c

la l-

:: t!?

=1.Tq l>t3 t;il.) l:lo l/)

l*,

--r Lt q!

- l'J .iL= !,l=-loL10 ltJ

= l/-\

l!.)o

a l=-

lo- lrDl-t*al>

ti ,-l- l-rr-- loo

hc:

a4=lQ =

=======

=

=

':a

=

-:-' l=

'; lr'

a-

LL^

Eqoa"

^- a.

-, lIt! 'L

t:

- \^ -1:

/- l\: G'lci =

:.t- rJ: t-\: lr!

CE

o-.)ta 7'

=-,:a=aJ!a,=

-

=====T

=

Figure 22

Chromaroqram of (a) Expired Spq!i!19Ql-4!dlo-Exp!rgd

Prednis-v-nth at 278 nm

12\

Table 42

Linear Regression Data and Validation Parameters ofPrednisolone Acetate aIId

Chloramphenicol tbr TLC Densitometry Method

I)nramrters Prednisolone Acetate Chloramphenicol

(at i.* 2,l3nm) (at;.,r 278 nm)

l.ine urit-,- llange 200-6000 ngispot 200-6000 ng/spot

(lorrolxtion Coolficient, 0.9966 10.035 0.992010.025

,.1SDY:mr+c 2.42x+1106 2.718x+4183

Sbpo 1SD 2.,1210.i 2.74 !0.34

lntcrccpt + SD 1106:0.48 4183 12.15

I'rrr.r-dn) (n=l),'7. RSD 1.1: 109

(nter-day (n=3), % RSD 1.90 1.68

t,imit of detection 0.0477 ng/spot 0.0300 ng/spot

Limit o f q uantilication 0.1,146 ng/spot 0-0912 ng/spot

Robust Robust

SpeciticSpc(ilicity Specific

726

Table 43

Summary of Stress Degradation Studies ofPrednisolone Acetate and Prednisolone

Acetate and Chloramphenicol in Combination

l)egratlation % Degrarlation yo D€gradatiofl in -Rl ofDegraded(-ondilions Combinxtion Products

,tciLlic Hydrolysis "

1N ltcl

5\ TI('l 100

t00 0.01,0.03,0.17,0.19,0.29,0.73

100 0.01.0.02.0.03.0.29. 0.58,0.78,0.80

lJ rsrr H\ drol\ sls

0.1\ \aOH 100 100 0.01,0.02.0.17,

L \ \aOH

iN NaOH

100

100

0.36,0.50,0.78r 00 0.01. 0.02. 0.01.

0.29,0.50100 0.01, 0.02, 0.29, 0.50

Nc utral t{,vdro I,vsis'

u.o 11.3 74.5 0.01,0.02,0.17,0.29

t1 0.01.0.17.0.26.0.29. 0.46. 0.'17- 0.80

0 0.01,0.17.0.26.0.1'7,0.'18

85.8 0.01.0.29.0.11.0.,17,0.58.0.73

\\'e! Hcatingn 95.9

I)ry' Heating 18.6

Photochcmical 100

OxidaIiLD

l59ov/v HrO:" 21 1

oxidalion at room 5.5

i0.6 0.05,0.14,0.29

9.0 0.05, 0.14, 0.29

temperatureb

"ttilux in parallel s) nthcsizer ior tu'o hours at 80 "C-

hkept tbr 24 hou$

127

Table 44

Sumrnary of Stress I)egradation Studies of Chloramphenicol and Prednisolone

Acctatc and Chloramphenicol in Combination

Degradation 7o Degradation 'llo Degradation in -Rr o{Degrad€dConditions Combination Products

,\cidic hydrolysis'

ti\ HCI

5\ IICI

23.8 I1.8

69 4 82.7

0.01,0.04

0.01,0.04,0.8i

llasic hydrollsis"

0.l N Naoll 100

l\ NaOH 100

5N NaOH 100

100

100

100

0.01. 0.44. 0.79

0.01,0.04,0.81

0.01,0.02,0.04.0.48

\cutlal hydrol,"-sis "

H,d 14.,1 17.9 0.01

0.01.0.79

0.01,0.79

0.01.0.09,0. r s.0.27,0.44,0.81 .0.85

ucr hcaling" 2tl.:I 36.9

2.1.0 0

Pr,l,".r',.lrr1""r 26.0 17.1

( )xidarion

-l5o o\ / \, HrO2' 7.)

13.4

0.01.0.12.0.31

0.01,0.12,0.31oxidrtion at room 9.6lcnlpel-a!LLreh

"Retlux in parallel synthesizer for two hours at 80 'C

bkept tbr 24 hours

128

2-2.2 l)examethasone, Chloramphenicol and Both in Combination

Literure survey revea ed thal simultaneous determination of Dexamethasone,

('hloramphenicol and a degraded prodr-Ict. 2- amino- 1-(,:l-nitrophenyl)propane- 1,3-diol in

ophthalmic solutions through HPLC is reponed. Fe* HPTLC methcds are also found

icported for the determinalion of Chloramphenicol in combination with various different

cornpounds separately like benzocaire and 2-amino- l -(,1-nitrophenyl) propane- 1,3-diol

(ANPD). and with differcnt coticosteroids including Dexamethasonesodium Phosphate.

llclamethasonc Sodium Phosphate. Prednisolone. Prednisolone Sodium Phosphate.

ll\'drocortisone. Hydroconisone Acetate and Betamethasone- 17-Valerate[@]. Simultaneous

determination of Dexamethasone and Chloramphenicol in the p.esence of their degraded

produots through I LC-densiiometry has not been reported so far-

Validated. simple ard accurate -l LC-densitomeiry method for the simultaneous detemination

ol l)cxamerhasone and Chloramphenicol in presence oftheir degraded products (by applying

stress conditions)\.!'as developed, following ICH guidelines Q1A in considemtion. As all lhe

pharmaceutical prodLLcts are supposed to be assayed for potenc-v, a validated TLC-

dcnsilomctry method. that do not show an.v inte 'erences of degraded products with the drug

active components is useful in measu ng these components in rolrtine analysis The

dcvclopcd I l.(' mcthod can be applied lor analysis of Dexamethasone and Chlor:unphenicol

in presence of their degraded producls in their individual and combined pharmaceutical

fbrmulations.Samples were spotted on HPTLC silica gel plates 60 F 254 and were deveioped

in chlorotbrm: methanol (14:l 2nd run). Aller development, Dexamethasone and

f hloramphenicol were found at Rr value of 0.181 0.02 and 0.21 l- 0.015, respectively-

729

Optimization 0f TLC Systcm and Mcthod Validation

Solvent system was optimized with standards, samples and degraded products. Diff0rcnl

solvent systens of acetone, methanol, 2-propanol, chloroform, toluene, ether and glacial

acotic acid were tried in varying ratios. Suitablc scparalion of activc componenls and

degraded products was achieved with chlorofora : methanol (14 : I v/v 2"d run) rvhich

showed sharp bands with l?r value of Dexamethasone at 0.18 1 0.02 and Chloramphenicol at

0.21 1 0.015, rospcctively.

Standard calibratior curve ofboth Dexamethasone and Chloramphcnicol was found lincar in

the coDcentration rangc of 200-6000 ng/spot with ,'71S.O. O.ggOe ! 0.035 and 0.9981 I

0.024, respectively. llor Intra-day and inter-day precision, '% RSD observed for

Dcxamethasone was 3.87 and 2.9) and, respectivcly while lor Chlolamphcnicol,2.l9 and

2.08, respectively. For Dexanethasone and Chloramphenicol, LOD wcre 0.09 nelspot and

0.03 ng/spot, rcspectively while LOQ were 0.27 ng/spot and 0.07 ng/spot, respectively. l'eak

purity was estimated by comparing the peak positions of both l)exanrethasone and

Chloramphenicol in stahdard speotra with those in reaction solutions. Good cor-relalion, 12

(start, nriddle) - 0.999 and ltniddle, end) = 0.999 was obsorvcd by comparing the spectra

of slandard and samples, in both cascs- The linear regtcssion data and lhe mcthod validation

paraDetcrs are summarized in Table 45.

Stabilily Indicating I'roperty in Dcxamcthasonc

Acidic, alkaline and neutral hydrolyses conditions, wet heat and pholoohcmical conditions

showcd greater degradation effects to Dexamethasone. Under various strcss conditions with

some common and diflerent peaks, total nine degraded products werc obscNcd. Peaks with

lowcr -nrvalucs indicated high polar nature than Dexamcthasone.

130

.lcilic llytlrolysis

ln 1N HCl, Dexamethasone was decomposed up to 45.7 % while ;n 5N llcl, it decomposed

upto 68.47o. Acommon peak at .llf 0.03 was generated under both the acidic conditions while

three additional peaks were also geneatrated under strong acidic condition at /lf 0.43, 0.70

and 0.83.

Alkaline II!.lrolysis

100% dcgradation was obscrved under alkalinc conditions. Common dceraded peak was

obscrvcd at.Ri 0.03. In addition to common peak, in 0.lN NaOlJ, an additional pcak was

also observed at 11r 0.62.

Neubal I-Iy.lrob)sis

Undcr neutral hydrolysis conditions, Dexamcthasone was lound stable.

lYel a d Dt! Ileal Degiadoliotl

Dcxanlelhasone showcd 42.5 % and 0.5 % degradat;on under wct heal and dry hoat

deSradation conditions, respectively. Both conditions showed common degraded peaks al Rr

0.03 while additional peaks were obseryed at Rr0.43 and 0.70 lor wet heating conditions.

Oxidatioh Reaclion

Onc of the oxidatiorl rrixlurcs was rcfluxed for 2 hours at 80 'C wllile the second was kcpt

lor 24 hours at room temperatute, Dexanrethasone was found stablc Llndcr both thc

coDditions.

Pholo Degradation Cohtliliot s

28.1% degradalion was observed under photo degradation conditions:nd seven degradcd

peaks wcre generated at 1+ 0.03, 0.33, 0.37, 0.43, 0.62, 0.70 and 0.83.

131

..\ common degraded product \,vith Rt 0.03 was generated under all stress conditions except

oxidation redction and neutral hydrolysis. Peaks at lir 0.43and 0.70 were commonly found

under acidic (5N HCI), wet heat and photo degradation conditions. A common peak rt RP.62

was generated under alkaline hydrolysis (0.lN NaOH) and photo degladation.Peak at Rr 0.8.1

uas tbund in stress conditions hcluding acidic (5N HCI) and photo degradation. T\r'o

additional peaks at R1 0.13 and 0.37 $ere on1) generated under photo degradation conditions-

Stability Indicating Property in Chloramphenicol

( hloramphenicol sho\r'ed greater susceptibiliry

and photo degradation stress conditions.

Aci.lic H!,dru|lsis

to degradation under alkaline. wet heating

ln lN HCl and 5N Hcl.Chloramphenicol rvas degraded upto2l 27o and71 0 9/i Common

dcgrdcd peaks rvere observed at nr 0.01 and 0.04. u'hile in strong acidic medium (5N HCI)

ar acldiLional peek at Rr 0.81rl,as also generated.

.llkdine Ilydr oll,sis

ChLoramphenicol was I00 7o degraded under all aikaiine conditions and a common degraded

pcak at Rl 0.01 was generated Under 0.1 N NaOH aikaline conditions' two additional

degraded products were obseNed at -Rr 0.215 and 0.79' while peaks at RO 04 was commonly

gcnerated under 0.1 N and 5N NaOH. Peak ai RO.81 was also found in lN NaOH treated

solntion. Trvo additionai peaks other than the common peaks were also generated at Rf 0 02

and 0.48 in 5N NaOLI treated solution.

Neutrd Il)jdrol))sis

Iinder oeutml hydrollsis. Chloramphenicol rvas degraded upto 13 9 % and a single degraded

product was tbund at -R;0.01.

732

Wel lleal and Dry lleal Degrudalion

'l'wo common additional pcaks were found in wet and dty heat dogradation conditions al-1lr

0.01 and 0.79 and dcgradation was upto 31.2% u.der wet heat and 19.8% under dry hcar.

()xidalion Reaclions

tlnder both the oxidation co'rditions, three additional peaks at,4f 0.01, 0.14 and 0.31 were

Sencraled and de8r,rdatron was les5lhall l) oo,

l' lto toc he m ic aI R e ac I io n

UDdcr photochcmical dcgradation conditions, scvcn additional p€aks at Rr 0.01, 0.09, 0.14,

0.27, 0.45, 0.81 and 0.85 werc generated and degradation was loss than 30 7n.

Under all stressed conditions, degraded product with -Rr 0.01 was commonly found whilc

desraded pcak wifi ,4r 0.02 was only observed undcr alkaline hydrolysis (5N NaOII). Peak

with /li 0.04 was gcnoratcd by acidic (l and 5N HCI) and alkaline hydrolysis (l and 5N

NaOH), while peak at.R10.48 was observed only in alkaline hydrolysis (5N NaOLI). tJndcr

alkaline hydrolysis (0.lN NaOH) and photo d egradatio 11_ reactio n s, pcak wilh tf 0 45 w,s

gcncraled. Degmdcd producls uilh ,4r 0.14 $,ls cotntrrorrl) iounLl Lrrrdcr ox;tlali,'rr rr'd Plrolo

dcgradation conditions while peak at lr 0.31 was only found under oxidution reaction.

Dcgraded peak with .11.0.79 was gencrated by alkaline hydrolysis (0.1N Naoll) and wet and

dry heat degradations. Under acidic hydrolysis (5N HCI), alkaline hydrolysis (1N NaOll) and

photo degradation corditions, degraded product with i?r 0.81 was gencrated. Peaks with laf

0.09, 0.27 and 0.85 were only generated through photo degradation roaotion. Video

densitogram piclures are showh ;n Iig rc 23.

Stability Indicaling Propcray o[ I)cxamcthas{rnc and Chlornmphcnicol in

Comhination

133

Acidic llydrollsis

Dexamcthasone and Chloramphcnicol were degraded upto 5'7.1 oA and 33.4 % undcr mil.l

acidic conditions whilc 69.2 % and 85.2 % undcr strong acidic condilions, rcspcclivoly. In

comparison with thc individual standard slress conditions od D€xarncthasone, a combined

standard solution showed an additional p€ak undcr acidic (lN IlCl) condition at /?r 0.E1,

corresponds 10 the degraded product ofChloramphenicol.

Alkaline Ilydrolysis

Dcxanrclhasone and Chlorarrphenicol were conlplelcly degraded under alkaline conditions.

A common additional peak was generalcd Lrnder all alkalirc condilions at Rr 0.02. Under

0.lN NaOH stress condition, Chloramphenicol degraded peak at -llr 045 was missing.

Moreover. undcr lN Naorl stress condition, an additional peak was obscrvcd at l?r0.45 rvhilc

Chlolamphcnicol deSraded products at & 0.04 was nrissing. In 5N NaOII trcalcd solution'

comparison showed Chloramphenicol degraded prodttcts peaks at -R10.04 and 0.48, rlrissing.

Neutal IIvfuolysb

In neutral hydrolysis roaction mixture an additional pcak for dcgraded prodllcl of

Chloramphcnicol was gcncratcd at L10.79.

Wel dnd Dry lleal Degradation

tJnder wct heat degradat;on coodition, Dcxamethasone was 51.2 % degradcd whilc

Chloramphenicol was degraded up to 37.1 %. Undcr dry heat dcgladatioD condition,

Dcxamcthasonc was stablc whilc Chloramplrcnicol was dcgrndcd up to 1.7%

134

oxidali?t, Reaclion

For oxidation reaction, two conditions were applied. LJnder oxidation condition (refluxed fbr

two hours at 80'C), Chloramphcnicol was desraded up to 8.1 % whilc under sccond

oxidation condition (roaction mixture kept for 24 hours at room tcmperature). Dogradation %

was 14.3 %. Under both the conditions, Dexamethasone rcmaincd stable.

P h ol oc lt e mic al D e g r adalio n C o n (l it io n s

LJtclor photochemical dcgradalion condition. pcal<s lor dcgradcd proLlucrs of

Chlorarnphenicol at1?r0.09, 0.14, 0.27, 0.45, 0.81 and 0.85 wcrc missine.

Stress degradation study ofDexamethasone ana Cnto.o-ph.ni"or ln individual and combined

sanrples is summarized in Table 46 and 47 while video densitogram picture is shown in

Fieure 23.

Analysis of Markctcd Samplcs

'l.wo expired pharmacculioal products were also analyzod- ln expircd sanrple (spersinicot).

pcak was not lourd for Chloramph€nicol at nf 0.2t (t00 % degradation) but six degradcd

poaks wcrc found at 1+ 0.01, 0.09, 0.14, 0.19, 0.38 and 0.58. ln expircd dcxopric-C prodlrcr,

peak for Chloramphcnicol was found but with 73 9/. degradation and Dexamethasone was

degraded up to 78 o%. Five additional poaks were also found at .Rf 0.01, 0.14, 0.38, 0.45 and

0.85. Comparison showed that degraded product at ,R10.01 product in boih,

nrdividual and ir cornbinarion. Degraded products at 11.0.14 and 0.38 were gcocrated by thc

dogradation of Chloranrphenicol. Degradcd pr.oducts with /?f 0.45 and 0.85 werc associated

wilh the degradation of Dexamethasone.

135

Figure 23

TLC Plate Showine Soots ofStandard Chloramohenicol, Dexamethasone.

Chloramphenicol and Dexame@NaOH" lN NaOH. 5N NaOH. Neutral Hvdrolysis. Oxidation and Photo Desradation

136

-

'l'ablc 45

l-ircar l{egressi<u l)ala and Validation l)alaltetcrs ol ])cxamcthast.rtrc ancl

Chlorarnphenicol for 1'[.C Dcrsitometry Method

l'arametcrs Dextmcthnsone Chlttramphcnicol

(at r,,", 238.5nrn) (at;,,,r, 27tt nm)

Linorrity llangc

Corrcla(ion Cocflicient, 0.996610.0i5 0.99811 0.024

121SD

Y=mx*c 1.299x + 27ll 2.72x r 4185

200 6000 ng/spot 200-6000 ng/spot

r.299 I 0.04 2.12 Lo t5

Intcrccpt ISD 27ll !0.46 418512.08

Siopc i sl)

Intra-day (n=3), % RSD 3.87

blct<lay (r:3), % RSD 2.91

2. 19

Limi( ot dctcction 0 09 n8/sPot 0 01 rrg/spor

l.irnil otquantificalion U.l. ng s|'ut l'.r|r rrs f'L

2.08

SpecillcSpecificity Specific

1-31

'I'able 46

Summary of Stress Degradation Studies ofDexamethasonc- and Dexamethasonc and

Chloramphenicol in Combination

Dcgradatior %o Dcgradat'ion 9/o Dcgradation in 1ir oI l)cgradod l,roductsConditions

n crdrc llydrolysrs '

Combination

!N IIC1

5N HCI

45.1

68.4

57 .1

69.2

0.01

0.03,0.43,0.70,0.83

13asio Hydrolysis

0.1N NaOII

lN NaOil

5N NaOH

0.03,0.62

0.03

0.03

t00 100

r00

100

100

100

Neutral Flydrolysis a

H70 0

51.2

0

Wct HeatiDg " 42.5

Dry Heating 0.5

0.03,0.41,0.70

0.03

I'lrotochemical 28.1 78.3 0.03,0.33,0.37,0.41,0.62,0.70,0.83

Oxidalior

35%v/v [IrOr' 0

Oxidatior at room 0

Reflux in paraUel syDthcsizer for two hours at 80 'C

okept for 24 hours

133

Tah)e 47

Summary olStress Degradation Studies of Chloramphenicoi, and I)cxamethasonc and

Chloramphcnicol in Cotrbination

l)cgradation 7o Dcgradation yo Degradation Ill olDegradcd ProductsColditions in CombinalionA.cidic hydrolysis "

lN fICt 2t.2 33.4 0.01,0.04

5N HCI 11.0 85.2 0.01,0.04,0.81

o.lNNaolr loo too o.ot,0.45,0.79

, INNaOII 100 100 0.01,0.04,0.81

5N NaOH 100 100 0.01, 0.02, 0.04, 0.48

Neutral hydrolysis'

'1tl)

Wct heating' 31.2 37.1 0.01, 0.79

lq 8 1,7 0.01.0.79llry heating

pt oioihimical - Xrt it.e 0.01,0.0q,0.14,0.27,0.i1,0.45,0.81,0.85

oxidaiion

irt"v/v H,O, e 0 01.0.14,0.11).1 b.t

Oxidation at room 11.2 14.3 0.01,0.14,0.31Iem perallue* I{ci1ux il] palallcl synthesizer for two hours al 80 'C

bkopl lor 24 hours

139

2.2.3 l)examcthasone, Prcdnisolonc Acctatc and Chloramphcnicol in

Combination

Litcrurc survcy rcvcalcd that fcw FIP l'LC rnethods arc rcportcd for thc dctcrmination (]1'

Chloraurphenicol in combination with various different compounds scparatcly likc bcnzooainc

and 2-amino- l -(4-nitrophenyl) propanc- 1 ,3-diol (ANPD), and with diffcrcrt cortioostcroids

including Prednisolone, Prednisolone Sodium Phosphate, Dexamethasone Sodium l'hosphatc,

llctiuncthasone Sodium I'hosphate, I{ydrocortisonc, Ilydrocortisonc Acctatc and Bclamcthasonc

I 7-Valeratet6al. Simultaneous determination of Dexanicthasonc. Prcdnisolonc Acetatc and

Chloramphenicol in presence oftheir degraded products through TLC-deDsitometry has rlot bccD

rcportcd so far.

Validatcd, simplc and accuralc:l-LC dcnsiiomctry mcthod lbr thc siurutanoous dclonninatiol1 o1'

two corticosteroid, Dexamethasoie, Prednisolone Acetate and an antibiotic, Chloramphenicol ir,

prescncc of their degraded products (by applying stress conditions) was developcd, following

ICIJ guidelines QIA ill considemtion. As all the pharrnaccutical products are supposcd 1o be

assaycd for potency, a validated Tl-C-densitomefly method, that do not show aDy irltcti-crcnccs

oI dcgraded prodncts with the drug active componerlts is useful in mcasuring thcsc componcnts

in routiDe analysis. Thc developcd fl-C method can bc uscd for routinc analysis o1'

Dcxamethasone, Prednisolonc Acctatc and Chloramphenicol in prescucc ol thcil dcgladcd

producls in their individual and cornbiDed plurmaceutical formulatious.

Optimization of l'LC Systcm and Mothod Yalidation

Solvent system was optimized with standards, samples altd dcgradcd proclucts. Differelt solvent

systems ofacctone, methanol, 2-propanol, ihloroform, toluerc, cther and glacial acolic acid wcrc

140

t 0d il1 varying ratios. Most of the solvent systems showcd diffirsecl spots of l,rcdnisoloDc

Aoctatc. Suitable separalion with best resolution was achievcd with chloroform : rnelhanol (14 :

I v/v 2"d run) which showcd sharp bands with ,1lr value of ChloramphcDicol at O.2l ! 0.02,

Dexamelhasore at 0.18 :! 0.02 and of Prednisolone Acetate at 0.41 -! 0.03. Stanclard caliblatioo

curye ol Dexamethasoie, Prednisolonc Acctalc and ChloEmphcllicol was ft)und linclr iu thc

conceirrarion range of 200-6000 ng/spor with 12 -r S.D. 0.9966 ! 0.029,0.99119 q 0.033 and

0.9920 ! 0.024, respectively. For Inka-day ard inter-day precision, '% RSD observed lbr

l)cxamcthasoDe was 2.19 and 4.01, 3.43 and 1.20 for Prednisolouc Acetatc whilc lor

ahloramphonicol, 1.18 and 2.06, respectively. For DexarncthasoDc, Prcddsolone Acclate and

Cirloramphenicol, LOD werc 0.07 ng/spot,0.04 ng/spot and 0.03 ng/spot rcspcctively while

l,OQ wcrc 0.22 ng/spol,0.14 ng/spot aud 0.09 ng/spot, rcsPcclivcly. l'oak plrrity was cstimatcd

by comparing the peak positions of Dexamethasonc. Pred solone Acctatc and Chlolamphenicol

in stJn.ldrd spectr.i wrllr lhosc ln rcactlon solution' Cood conel'rlr"rr' r" rslrrl' mid'll( r n '^")

alld r7 (middle, end) - 0.999 was observed by comparing the spcctra o[ standlrd and samplcs. itr

both cases. lhc lincar rcgression data aDd thc melhod validalion paramcters arc sumnrariTed in

Table 48.

Stability Indicating Property in Dexamethasone

Acidic, alkaliDe and Deutral hydrolyscs condilions, wet heat and photochemical oorditions

showcd groatcr dcgradation olfccts lo l]cxalnclhasottc. l]ndcr various strcss couditions with

some commorl a11d different peaks, totai scvcn deBraded products *erc obsoNcd. Pcaks wilh

lower llrvalues indicatcd high polal nature thall Dexamcthasone

141

,lcidic l\)dt-oltsis

In lN HCl, Dcxametha-sone was decomfosed up to 40.1 ,. $hilc in 5N lICi, il dccomposed uplo

'12.9yn. A,n additional peak at /?i 0.03 was gcncrated ulder mild acidic condili.r.rs whilc lbru

additional peaks were gencatrated under stoDg acidic condition at 1lf 0.03 and 0.43, 0.70 and

t) 11r

Alkaline llydrofusis

100% degradation was obscrvcd under all alkaline conditiurs. I)egradcrl pcak was obscrved at /?1

0.01. In addition to common peak, in o.lN NaOH, an additional poak was also obscrvcd at 1(r

0.62.

Neulral Ilydrolltsis

ljnder ncutlal hydrolysis conditions, Dexamethasonc was lound stable and dcgradation was upto

1.9%,.

llet urd Dry lle Degrsdstion

Dcxamcthasone showed 43.4 % and 3.1 % degradatiol undcr wct hcal ard chy hcat dcgradation

conditions, respectivcly- lloth conditions showed comrnon degraded pcaks at /lf 0.03 whilc

additional peaks wcrc obscrved at 1?f 0.43 and 0.70 for wet lleating conditions.

Oxidalion Reqclion

Dcramcthr.,rrre was lounJ slablc under both the oxidatiol tonditiols.

142

I'hoto Degradalion Con.litions

31.5 oZ dcgradatiorl was observed uDder photochemical conditiors and scveD dcgmcled pcaks

wcrc geooratcd at 1if 0.03, 0.13, 0.17, 0.43, 0.62, 0.70 and 0.g j.

A corrmon degradcd producl wifi 1lr 0.03 was gencratcd under all slross cotldilions exccpl

oxidatiol rcaolion and neutal hydrolysis. Two additional peal$ at /l.0.33 aDd 0.37 werc o]lly

generated under photo dcgradation conditions. peaks at 1?s 0.43 and 0_70 werc corrmonly founcl

ulder acidic (5N IlCl), wet heat and photo degEdation corditions. A common pcak at,1110.62

was geierated undcr alkalinc hydrolysis (0.1N NaOII) and photo dcgradatioi i)cak al 1i 0.g3

was found itl stress conditions including acidic (5N HCI) aDd photo dcgradation ( I ablc 49).

Stability Indicating l,ropcrty in l,rcdnisolonc Acctatc

Acidic, alkali,c and neutral hydrolyses conditions, wct hcat and pllotocllenical conditions

sho*cd grcater dcgradation cflects to Prednisolone ,\celatc. ljnder-vario s slress conditions with

some common and diflercnt peaks, total eighteen degraded products wcrc observcd. pcaks r.vitir

lower 1?fvalLres indicated high polar nature than prednisolone Acetatc.

,lcirlic llydrolysis

hr lN IICI. Prednisolone Acet.ate was dcconposed up to 75.1 % whilc iD 5N IICI, it clecomposed

upto 100%. Three additional peaks at 1+0.01,0.03 and 0.29 wcrc conrDronly geDcratcd in bolh

acidic rcaction mixtutcs. In additiur to those peaks, only in lN lICl lcaclion mixtLu-c. throc

additional pcaks at .Rf 0.17, 0.19 and 0.?3 were also geDerrled while four ad(litionaj peaks ar Jlf

0.02, 0.58, 0.78 aDd 0.80 were only obtained under strong acidio condilion (5N IICl).

14.1

/lkaline llydtolysis

100% dcgradation was obscrved under alkaline conditions. Common clegracled pcaks were

obscrved at 1i1 0.01, 0.02 and 0.50. In additior to common pcaks, in 0.lN Naoll. thrcc additional

peaks were also observcd at nf 0.17, 0.j6 and 0.78, aud an additioral pcaks a1 11r0.29 in lN and

5N NaOII trcatcd solutions was also found. Peak a1 .(r 0.03 was orly gcncr.atcd in lN NaOII

(eated solution.

Neutral Ilydrolysis

\Under Deutral ludroll/sis conditions, four additional peaks at,Rr 0.01, 0.02, 0.17 aud 0.29 wcrc

generalcd and Predni.olone Acelalc uas degradcd uplo 7q 10,,.

Wel tuitl DrJr lleot Degtadation

Prednisolonc Aoetate showed 97.1Y. and 20.102 degradatioD uDdel wet hcat aud dry heat

dcgradatioD conditions, rcspectively. Both conditions showed common dcgmdcd pcaks at /lr

0.01, 0.17, 0.26 and 0.47, while additional pcaks werc obscrved at ltf 0.29, 0.46 and 0.80 tor wel

hcaling ard at 1lf 0.78 fo. dry heating condirions.

oxitlstiofi llesction

One ofthe oxidation mixtues was refluxed for 2 hours at 80 .C whilc the secord was kcpt fol 24

hours at room tempcralure aDd showed 21.|yu ar;1d 5.5r% degradalion, rcspcclivcly. Ljnclcr botlr

thc oxidation condilions, thrcc commoD pcaks were gencratcd at 11r 0.05, 0.14. and 0.29.

744

I' holo D egradatio n Co ntlitiq ns

i00% dcgmdation was obscrvcd unclcl photochcniical 0oldiliolN ard tlcgrarlccl pcahs rvcrc

gercrated at 1?r 0.01, 0.29, 0.33, 0.47, 0.58 and 0.73.

Urder all the stress conditions excepl oxidatioD rcaction, a commoD dcgraded Product with Ir

0.0i was gorcrated. Degraded product with lr 0 02 was only gereratcd by acidic (5N IICI),

alkaline (0.1, 1 and 5N NaOH) aDd rcutral hydrolvsis. Pcak at 1?r 0'03 was gcncrated Ddcr

acidio (l and 5N HCI) and alkaline (lN NaOH) hydtolysis whilc peaks at ,4r 0.05 alrd 0' 14 werc

only obtaincd by oxidation reactior. A.cidic 11N llCll. alkalinc (0'l N NaOll) ancl ncuhal

hldrolysis anJ wct lrcal ,rnd drl lleat dcgradation condllloll: (oll) llllll) g.rrclJI''l il 'lcgrJd''l

produot at 1?6 0.17, while peak at J?r 0.26 was oDly found by wct ancl clry hcaling conditions l'cak

at L1 0.29 was found in various strcss oonditiols inciuding acidic, alkaiinc (1 and 5N NaOH) and

ncutral hydlolysis, wet heatirg, oxidation and pLoto degradation. Charactelistic pcaks of photo

degradalion and allGliDe hydrolysis (0.1 N NaOII) rvcre found at llr 0 i3 iud 0.36, respcotivcly.

Similnrly peak at -iaf 0.46 was only generated in we1 heat degradalion reacliot. wl ilc a pcak wiih

/? 0.47 rvas obscrvcd in wet and dry ireati[g and photo dcgradation conc]itions. Dcgradcd prodtlct

at -1lr 0.58 was generated by acidic hydrolysis (5N IlCl) and photo dcgradation. l'eak with /lr 0.73

was found in acidic hydrolysis (lN HCI) and photo degradalion. Acidic hydrolysis (5N fl(-'l).

alkaline hydroiysis (0.1N NaOH) and dry heating commonly generatcd a dcgradcd poak at /?r

0.78. Degmdcd product a1 1?f 0.130 was generated through acidio hydrolysis (5N IICI) and wcl

hcat degradalion (Table 50).

Stability lndicating Prop€rty in Chloramphenicol

Chloramphenicol showed greatcr susceptibility to degradation to alkaline. wet heating and

pholochemical strcss conditions.

lcidic Hytbolysis

In lN HCI and 5N Hcl,Chloramphenicol was degraded upto24 6 9/r and6A 4 o/' Common

degraded peaks were observed at -Rr 0 01 and 0.04, while in strong acidic medium (5N HCI) an

dddrliundl pcdl \ a" generared cl Rr0.81.

Alkttlihe Hydrolysis

Chloramphenicol was 100 % dcgraded under all alkaline conditions and a common desraded

peak at Rr 0.01 was generated. Under 0 l N NaOH alkaline conditions' additional degraded

products were obscrved at nr 0.44 and 0.79, while peaks dt 'l?O 04 and 0 81 were also found in

lNNaol.ltreatedsolution.ThreeadditionalpeaksatRr0.02,0.04and0.48werefoundin5N

NaOI-l treated solution.

Neutral llydrolysis

Under ncutral hydrotysis a single degraded product was found at Rr 0.01 with 129%

degradation.

llet lleat and Dry Ileal Degrudalion

Two common additional peaks were found in wet and dry heat degradation conditions at nr 0 01

and 0.79.

746

Oxitldtion Redctions

t-lndcr both thc oxidation conditions (refluxing and at room tempemture lbr 24 hours), three

additional peaks at ,Rr 0.01, 0.14 and 0.3 1 were commonly generated with 5.4 and 1 0 58 %

degradation, r€spect;vely.

P lt oto c h e mical Re ^cti

on

tJndcr photochemicat degradation conditions, seven additional peaks at Rr0 o1,0 09,0'14,0 27,

{). I l, 0.ltl and 0.85 were generaled t{ith 2b.ooo dcgradalion

Urrrler all stressed conditions,degraded product with l?1 0.01 was commonly formed whilo

dc8radcd peak with nr 0.02 was only observed under alkaline hydrolysis (5N NaOH) Peak with

Rf 0.04 was generated by acidic (l and 5N HCI) and alkaline hydrolysis (l and 5N NaoH), while

pcak at nr0.48 was obscrved only in alkaline hydrolvsis (5N NaOII). l)egraded products with Rr

0.14 werc found under oxidation and photo degradation conditions, while peak with Rr 0 31 was

lomrcd oniy in oxidation reaction. Under alkaiine hydrolysis (0.1N NaOH) and photo

dcgradation reactions,peak with Rr 0.44 was generated Degraded peak with Rr 0'79 was

generated by alkaline hydrolysis (0.IN NaOH) and wet and dry heat degradations. Ilnder acidic

hydrolysis (5N HCI), atkaline hydrolysis (lN NaolI) and photo degradation conditions'

degraded prcduct with rtf 0.81 was generated. Similarly peaks with ,Rf 0 09, 0 27 and 0 85 tlcre

only gcnerated through photo degradation reaction (Table 51).

t47

Stability Inclicating Propcrly of ])examcthasonc, I'rcdnisolonc Acctirto and

Chlorarnphcnicol in Combination

lcitlic Ilylrolysis

ID co parison wilh the individual standard solutions, combined slandard soluliorr sho$'od lhrcc

clcgr-atlccl products of I'rcdnisolonc Acetate at /+ 0.03. 0.19 aud 0'73 missing while tr'vo

additiolal peaks turdcr acidic (lN llcl) conditioD at llr 0.78 and 0.81 wclc gcneratcd' Pcak at /lf

0-81 corcsponds to thc degraded product of Chlorar,rpheoicol four additional peaksat-Rr015,

0.38,0.56 an<l 0.62 were lound under 5N FICI slress condition while dcgraded produols of

Prcdnisolone Acetate with -Rr0.03 :rnd 0.80 were missing

,1 Ikaline Ilvdrol.ysis

Dexametlusore, Prcdnisolone Acetate and Chloramphenicol werc compictclv dcgradcd undcr

alkaliDo conditiorN. Undel al] alkaljnc conditiol'N, two comlnon additioml pcaks wctc genclatcd

ai 1lr 0.01 and 0.02. Undcr 0.1N NaOII strcss conditioD, 1wo additiorral pcaks werc found at .1?,

0.29 and 0.1i5 wl le Dexamethasone and Prednisolone Acctatc dcgradod peaks at 1if 0.16 and

0.78 and Chloran, phcnicol degraded peak at,tr0.44 were missing. Morcover, undcr lN NaOII

stress ooDditiorl, all additional peak was observed at nf0.44 while Chloranphcnicol dcgradcd

products at 1?1.0.04 and 0.81 and Preduisolone Acctate degraded product at 1lr 0.50 \ 'crc missing.

In 5N NaOII treatcd solutiorl, comparison showcd an additifial pcak at ll1 0.31 whiic

Chlorarnphenicol degradcd products at.1?r0.04 and 0.48 and Prcdnisolonc Acetalc rlegradod

products at .1ii 0.29 and 0.50 were missiDg.

148

Neutrol IIldrolYsis

In neutrai hydrolysis reaction mixture, peak for Ptednisolone Acetate at -Rr 0 29 was missing and

an additional peak was gcnerated at Rr0'?9'

Wet dn,! Irry Haat Degtadatirtn

An addilional degraded product at Rr 0 50 was observed in combined sample under wet heat

degradation while degraded products ofPrednisolone Acetate at RP 26' 0 46' 0 47 and 0 80 were

missing- Undel wet heat degradation condition' Dexemethasone was degraded up to 38%'

l,rerlnisolone Acetate 1770 whilc Chloramphenicol was degraded up to 36.9 7o. Under dry heat

degr-adation condilion, comparison showed an additional peak at Rt 0 73 while Prednisolone

Acctate degmded peaks at,Rr 0 17 and 0 78 were missing'

Oxfulation Reaction

For oxidatior reaclion, two conditions were applied'Under oxidation condition (refluxcd for two

hours al Il0 "C), an additionai peak at -Rr 0 55 was observed while two additional peaks at nr() 23

and 0.55 u'ere observed under second oxidation condition (rcaction mixture kept for 24 hours al

rtxnn tcmperature). Prednisolone Acetate degraded peaks at Rr0 05' 0 14 and 0 29 were missing'

P h r t ot h e mi cal Degratl at i on Condit ions

Under photochemical degradation condition, additional degraded products at R049 0'50 and

0.69 r,(rr(j Eeneratcd only in combined sairple while a degradcd product of l'examcthasone'

Ptednisolone Acetatc at Rt 0'29 arld 0.33 and thosg of Chlotamphenicol at fif 0.09, 0' 15, 0.28,

0.,14.0.81 and 0.85 were rnissing'

149

Stless deSradation study of Dexamethasone, Prednisolone Acetate and Chloramphenicol in

individual and combined samples is summarized jn Table 49 -5lwhile video densitogtam

pictures are shown in F'igute 24.

Slrcss degradation study data showed that under all stress conditions, peak with Rr 0'01 was

comnonly fonncd. Degraded produots with Ri 0.09 and 0 15 were formed by photodegrdation'

Degraded products with Rr 0.38 and 0.58 wete not found under any stress condition applied to

Chloramphcnicol alone. Dcgraderl product w;th 1lr 0 38 was Eenerated in combincd sample of

I)crarnothasone, Ptednisolone Acetatc and chloramphenicol under acidic hydrolysis (5N HCI)'

150

Figtre 24

TLC Plate Showins Spots of Standard Dexamethasone. Chlonmphenicol. Prednisolone

Acetate. Acidic Hydrolysis. Alkaline Hydrolysis. Neutral Hvdrolvsis. Wet Heat

Deeradation and Photo Deqradation

151

(D ID

l'able 48

Linear Regrcssion Data and Validation Paramete$ ofDcxamethasone, PrcclnisoloneAcetatc ard Chloramphenicol for TLC Densitornctry Method

Prcdnisolon(. Ar( lat; -irn.r-pt ".i"or(41.1,",- 243 nm) (at,i,,,,r 278 nm)

Dcxamethasonc(rt 2,,,", 2311.5 nm)

Lilrcarit] Ilange

CorrclationCocfficicnt,,'+ sl)

Skrpc 1SD

lnlerccpt t SD

Intraiay (n=3), %Itsl)

rntcr-day (n=3)r %IISI)

Limit ol dctcrtion

l,imit ol qurnlilic:rti(,n

Iiobrstncss

spcci{icit)

200 6000 ng/spot

0.9966 t 0.029

1.299x12711

1 .299 !05

2',1t| 10.41

2.19

4.01

0.07 ng/spot

0.22 ng/spot

Robust

Specific

200-6000 ng/spot

0.9989:l'0.033

2A2x I l106

2.42 xO.5

I10610.48

3.43

200-6000 ug/spot

0.9920.r 0.024

2.7313x I ,ll83

2.738 !0.34

4183 12.15

Ll8

),.061.20

0.04 ng/spot

0.14 ug/spot

RobUSt

0.01 Dg/spol

0.09 ng/spot

Robust

Specific Spccilic

152

Table 49

Sul]r[raly of Stless L)egradation Studics ofDcxamethasone and Dexamethasone,

I\'cdnisolonc Acctatc and Chloramphenicol in Combination

Degradation 7o Degrtdation 9/o Dcgradation in ltrofDegradedConditions Combination l'roducts

Acidic ltydroiyiis i --

IN HCI

5N HCI

rl"";c irydrolyiis'-

40. i 59.1

72.9 71.8

0.03

0.03,0.43,0.70,0.83

o.lN NaOH 100 100

100

100

0.03,0.62

0.03

0.03

lN NaOH 100

5N NaOH 100

NeLrtrel Hydrolysiso

lt,o 1.9 1.1

wcr I lcalingu 43.4 55.4

i)ry lleating :i.1 1.2

0.03

0.03,0.43,0.70

0.03

Photochernical 31.5 81.8 0.03,0.33,0.37,0.43,0.62,0.70,0.84

Oxidation

j's'/"vlv H2O2' 0 0

0Oxidation af room 0ternpcratureb

'lleflux in parallel syrlthcsizer for two hours at 80 'C

bl(cpt fbr 24 hoLrrs

. 153

Table 50

Summary of Stress Degmdation Studies ofPrednisolone Acetate and Dexamethasone,

I'rednisolone Acetate and Chloramphenicol in Combination

frogradation 9/o Degradation 9/o Degradation in -RrofDegradedConditions

Aoidic Hydrolysis'

Combination Products

1N IICI

5N HCI

75.t

100

100

100

0.01,0.03,0.17,0.19,0.29,0.'130.01, 0.02, 0.03, 0.29,0.58,0.78,0.80

Uasic Hydrolysis'

0.lN NaOH 100 100

100

100

0.01, 0.02, 0.17, 0.36,0.s0,0.780.01,0.02,0.03,0.29,0.500.01,0.02,0.29,0.50

lN Naoll

- 5N NaOH

100

r00

Neutlal Ilydrolysis

'70 I 75.8

29

2.4

0.01, 0.02, 0. 17. 0.29

0.01, 0. 1'1, 0.26. 0.29,0.46,0.47.0.800.01,0.17,0.26,0.47,0.78

Wel Heating' 97.1

l)ry Ileating 20.1

Photochcmical 100 9l .2 0.01, 0.29, 0.33, 0.47,0.58,0.73

Oxidation

i5%v/v Hror' 21.0s

oxidation at room 5.41tctnpelalurc

34.2

13.4

0.05,0.14,0.29

0.05,0.14,0.29

'llcllux in parallel synthesizer lor Iwo hours at 80 'C

bkept for 24 houls

754

Table 5 1

Sumurary of Stress Degradation Studies of Chloramphenicol and Dexamethasone,l'rednisolone Acctate and Chloramphenicol in Combination

Degradation 7o Degradation g/" Degradation in XrofDegradedConditions Combination Products

,tciaic nyarotl,s;s'

IN IICI

5N HCI

2.4.6 33.8

68.4 76.2

0.01,0.04

0.01, 0.04, 0.81

Basic hydrolysis"

o.tN NaoH loo

lN Naoll 100

5N NaOH 100

100

100

100

0.01,0.4s, 0.79

0.01,0.04,0.81

0.01, 0.02, 0.04, 0.48

Ncrrtral Ir1'drolysis "

llrO 0.01

0.01,0.79

0.01,0.79

\2.9

wcr heating" 32.6 41.9

21.8 0Dry heating

18. I

I)hotochemical 26.6 25.4 0.01, 0.09, 0. 14, 0.27,0.31, O.45,0.81,0.85

Oxidatior

l5%v/v IIzOz' 5.4 8.4

OxidatioD at room 10.58 I7.80.0t,0.14,0.310.01,0.14,0.31

temperaturJ

"llellux in parallel synthesizer for two hours at 80 "C

okcpt for 24 hours

155

3.0 EXPERIMENTAL

156

3.1 Calibration Standards and Sample Prcparation

Standards, Chioramphenicol, Dexarnethasone and Plednisolonc Acctalc wcrc complemcntarily

provided by Santa (Pvt.) Ltd, Karacli, Pakistan. I'harmacculical products including

Dcxarnethasone eye drops (D1, Ophthadcx, Ophtha Plurma; D2, Metludex, Jaens; D3,

Spersadex, Novarlis; D4, Bi[adex, Barette Hodgson) and Dexamethasone tablet (l)5,

Dexamethasone, Hamaz), Prednisolone Acetate eye drops (P1, Ophtha Pred, Schazoo; P2, Mildo

Pred, Remington; P3, Predforte, Barrette Hodgson; P4, Pred+, Schazoo; P5, Prcns, Vega),

Chloramphcnicol eye drops (C1, spcrsinico[, Novartis;-C2. chIrruptic, I]arcttc Ilodgsor),

capsule (C3, chlormycetin, Pfizer) and ointnent (C4, optachlor, Remiigton), and ophtbalmic

suspensions containing Dexamethasone and Chloramphenicol in combimtion (DC1, i)cxoptjc-(1,

' Slhuroo; DC2, fluorbioptal, Remington) Prednisolone Acetate .rDd Ohloramphcllicol in

oombination (PCl, prednisynth, Schazoo; l'C2, prcdnicol, RcmingtoD) wilh thc thlee expircd

pha naceutical products (ECl, spersinicol, Novartis; EDCI, dexoptic-C, Schazoo; EPCI,

orcddsynth, Schazoo) were purchased from local pharmacy shop, Karachi, I'akistao'

Mcthanol, acetonitrilo and chloroform of amlytical gmde wete purchascd fronr thc l.rsh(r

Scicntific (UK). Dei(xrizcd water was obtained lron Milliporc Milli Q l'lus Svstcn (t3edltld'

USA). Sodium hydroxide was purchascd fronl BioM Laboratories (Cerritos' IJSA) whilc

hydrocl o c acid (HCl) and hydrogcn peroxidc (HuOz, 357" v/v) wcrc ohlaincd fiorr |ishcr

Scientific (UK).

liivc sto0k solutions wcrc prcpared by dissolving 100 mg each of Dcxamothasone, I'rcdnisolonc

Acetate and Chloramphenicol in 100 mL methanol, individually and ir, combination' Working

standard solutions wer.e prcpaed by dilution of stock solutioD with methaDol to give solutions jn

1\1

conocntra(iol1 range of 50-1000 pglnl, for calibration curyeoflJltra Fast HPLC methods.

l)ilLrtion ofstock solution with methanol wxs done to prepare solutions in concentration range of

-10 I000 pg/ml for calibration curveofHPTLC methods, six-point calibration curve was formed

by spotiing 6 pl- ofeach standard solution in concentration mnge of200-6000 nglspot containing

both components in combination, each concentration was sPotted lhrice on six replicate plates'

Prednisolonc AcctatePharmaceutical Products

l'or sdmple preparation, I mL ophthalmic samples (P1, P2, P3,P4and P5) containing l0 mg of

Prednisolone Acctate were diluted in 20 mL volumetric flasks with methanol, separately'

DcxamcthasotrePharmaceutical Products

l.or sample prcparation, I mL ophthalmic samples (Dl, D2, D3 and D4) containing 10 mg of

Dexamethasonewercdilutedin20mLvolumetricflaskswithmethanol,separately.For

Dexamethasone tablet, 20 tablets were weighed on electronic balance, crushed and average of it'

0.1956 g was dissolvcd in lOrnI- ofrnethanol

Chloramphenicoll'harmaceutical Products

Sirnilarly, I mL ofspersinico, and chloroptic containing 0 5 % Chloramphenicol was diluted in

l0ml-volumetricflaskswithmetharol.Chloramphenicolcapsules,equivalentto2500mg

(chlormycctin) were placed in 500 mL of volumetric flask and after addition of 100 mL water'

heated on steam bath till the capsule was disintegrated After further addition of300 mL water it

was again heated on steam bath with mixing After cooling to room temperature, it was diluted to

volume with water. 5 mL of the resulting solution was transferred to 100 mL volumetric flasks

arrd diluted with mcthanol. Chloramphenicol ointment equivalent to 25 mg of Chloramphenicol

158

was transfe[ed to 100 mL volumetric flasks aDd diluted. with methano]. Chlorzurpheniool

ointmeut equivalcrt to 25 mg of Chloramphenicol was transfered in conical flask and aftcr

addition of 20 ml- of cyclohexane, mixed and sonicated for two mirutes. Futher 60 ml- of

mcthanol was added to this solution, mixed and then fiitercd. Filtlate was collectcd in 100 rnl-

volumclric Ilask, filter was also washed with methanol and washings wcrc collcclcd and vohuc

was nrade up wiih methanol. 50 mL of this solution was translerred in rotnd bottoln llask and

was dricd by rotating undq vacultm on water bath at 35 oC. Itesidue was dissolvcd iD 50 mL

mctl'ranol. 10 nl ol lhe resulting soiution was transferred in 25 mL volurncllio llask, dilrtcd with

methanol. Ophthalmic suspension oquivalent to 5mg of Prednisolone Acetate and 2 mg

Chlorirmpl, enicol was dissolved by sonication in an amount of methanol a1ld volllme was made

up with 20 mL melharol. I mL ophtbalmic samPles (plednisyDth and prednicol) containing 5 ir8

ol prcdnisolonc Acetate and 2 mg Chloramphenicol wcre diluted in 20 ml, volumetlic llask with

mclhauol.

i'harmaccutical l'roducts Containing Prcdnisolonc Acctatc and ChloramphcBicol,

and l)cxamcthasonc and Chlorarr phcnicol in Combination

I mL ophthalmic samples (Dexoplic-C and Fluorbioptal) containing 5 mg ofDexamcthasonc and

2 ng Chloramphenicol were dilutcd in 10 ffL volumetric flask with tnethanol'

1 mL ophthalmic samplcs (Plednisynth and Prednicol) conlaining 5 mg of l'rednisolonc Acotalc

dnd 1/2 g Chloramphenicol wcre diluted in 10 mL vollunctric flask willr mclhanol'

In similar marner, solutioDs for expired spersinicol, dexoptic C and prednisy[th were also

prcpared by diiution with methanol (lml. in 10 mL volnmetrio flask) All thc solulions wcrc

filtered with 0.45irm PTFII lluoropore syringe dtiven filtcr, urrit (Millip"re. Jlctlford, USA)'

3.2 Ultra-Fast HPLC Instrumentation and Parameters, Method Optimization

and Validation

Ultra Fast HPLC method was developed on Agilent l20O Series Rapid Resolution LC (RPLC)

system equipped with Agilent Binary Pump SL assembled with Degasser, High performance

aulosampler SL with thermostat, thennostataed collrmn compartment(TCC), Agilent Zorbex

Eclipse XD]S C-t 8 Column (50 x 4.6 mm I.D., 1.8 !.m) and diode array detector SL (DAD SL)'

(-hromatographic chcmstation software by Agilent Technologies was used for data processing

and integlation. lnjection volume was I pl while column temperature was set at 30'C Analysis

was done at multi wavelength of 254 nm, 243nm and 278nm through DAD detector and EI-SD

dcrcclor.Method was developerJ with binary gradient system, solvent A was water whilc solvent

B was acetonitrile.

Stock standard solution was prepared by dissolving 100 mg of Chloramphenicol and

Prednisolone Acetate in 100 ml methanol. Calibration standards were prepared by diluting the

stock solution within linearity range of 5o-l000pg/ml Triplioate injections was made for each

concentration (50,100,200,400,600,800 and 1000 pg/ml) Calibration graph was obtained bv

plottjng peak arca against concentration Parametcrs considered for optimization of

chromatographic systen included mobile phase composition, flow rate' sample volume' column

tcmperature and detcction wavelength. ln order to demonshate the suitability and reliability oi

dcvcloped method, it was validated as per requirements of ICH guidelines Q2A and Q2B'

l,incarity

'Ihc linearity (correlation coefficient) was determined by applying tliplicate injections of

srandard at sever dilfcrent concentration levels ranging from 50-1000pg/ml- applyiflg triplicate

160

injections of each concentration (50,100,200,400,600,800 and 1000 pglmL) for both

Plednisolone Acetate and Chloramphenicol in combination'

Precision and AccuracY

ln ordcr to dcmonstrate accuracy and precision of method, repeatability test was done lntraday

prccision was evalualed by applying six rePlicate injections for each five concentration levcls of

standard solutions (100,200,400,600 and 1000 pg/mL) lntermediate precision was evaluated

through three replicate iniections of standard of same concentration on different davs (lnter day

precision).

Limit of Dcleclion and Limil oI Qll antificalion

In order to estimate signal to noise ratio at 3:l and l0:1 for limit of detection and Iimit of

quantification respectively, calibration curve was used'

llobustness and SPecificitY

Minor deliberate changes in flow rate, column temperature, wavelength and mobile phase were

done in order to check the robustness. All pararneters were changed in 150% at concentration

lcvels of 200, 400 and 600 pg/L. The specificity of the proposed method was verified by

overtaying the chromatogram of standards and pharmaceutical products Peak purity and _Rr of

ChlorrLmphenicol, Dexamethasone and Prednisolone Acetate in pharmaceutical products were

compared with those in standard.

161

llccovcry Studies

l)re-analyzed sal))ples were fortified with three known standard conccnttatiors (2570, 50%, ard

75% ofsample). AII drugs wore subjectcd to rccovcry sludics and Lhc rcoovcrcd an'rounlol addod

drugs was detclmilled.

A[alysis of Chloramphenicol, l)examcthasonc flnd l'rcdnisolone Acctatc samplcs

I'ol UPLC analysis 2 pl of Chloramphenicol eyedrops samples, 4 prl of Chloramphenicol capsulc

and 5ul of its oirtmr:nt, 2 pi of Prednisolone Acetate prepared samplcs, 1 pl of Dexamethasone

prepared samples and 0.5 pl of its tablet sample, while 2 |.rl ofprep;red drug s.unples conlaining

Prednisolone Acetate and Chlorarnphcnicol in combination and i lLl oI l)cxamcthasonc and

Cl oramphenicol sample containing both in comibination were uscd for alla ysis

3.3 ICII ltccommcndcd Strcss Dcgradation Studics

Prednisolonc Acctatc and Chloramphcnicol

Methanolic stock solution (1 mghnl-) olboth Prednisolone Acetate (set l) and Choramphcnicol

(sct 2) was prcparcd, scparatciy and jn combinalion (sct 3) to pcrlotm lorced dcgradation Studics

ir parallel syDthesizer (Smarl Start Synthesizer, Cher,r Speed Ltd., Switzerland) by refl[xing thc

reaction mixtures for two hours at 80 "C. St(ess degradation studics wcrc pcrlbrmcd by

maintaining uniform degradation conditions using parallel syDthcsizer (Snlart Sta{ Syrrthesizcr-

Chen speed Ltd.. Switzerland) including acidic, alkaline and ncutral hydrolysis, oxidalion and

wet heatiDg degradation. Oxidation at room tcmperature, photochemical a11d dry heatirg

degradation studies were also caried out. After the reactions wcre conpleted, all the solutions

werc preserved al 80 'C till analysis.

162

,lcitlic, Alkaline and Neutntl H))tlrolysis

For acidic hydrolysis, lN and 5N FICI were used, for alkaline hydrolysis, 0.1N, lN and 5N

NaOH werc used while for neutral hydrolysis Milli 'Q water was used. 3 tnl- of each

concentratior of acidic and alkaline solutions and Milli Q water were added into 3 mL (1

rng/ml-) stock solutions ofall threc sets.

Wel Ileal Degraddtion

'lo study wet heat degradation, 3 mL (1 mglnl-) of stock solution of each set I' set 2 and set 3

was sub.jectcd to degradation.

Oxidatiofi Reaction

Oxidation was carried out by adding I mL ofHzO: (35%o v/v) in 3 'InL

stock solution ofeach set'

All the resultant solutions were refluxed for two hours at 80 'C in parallel synthesiTer

Dry Heal Degradalion .

Dry heat degradation was conducted by taken standard Prednisolone Acetate and

Chloramphenicol and heated in oven at 90 'C for 4 hrs'

Photo Degrudation

ln order to evaluatc phoLochemical degradation of Prednisolone Acetate' Chlorampllenicol and

both in combination, stock solution of each sct was directly exposed to the sunlight for threc

days from 8 to 18 hrs at 30 l- 2 "C.

163

,\ll the solutions rvere p.cscrved at 80"C till analysis. Scpatation was dotc on TI-C glass platcs.

pre-coatod with silica gcl 60 f-254 rcing chlorolorm : ncthanol ( 14:l v/v). Averagc pcak alcas

o1-aolivc compoients wcle alralyzcd afler triplicatc analysis.

l)cxamclhasonc and Chloramphcnicol

Mettranolic stock solution (l mgrnl-) oI both Dexametbasone (se1 4) and Chloramphcnicol (scl

2) \\,'as prcpared, separalely and in conbilution (sct 5) to pcrform lorccd dcgradation sludics in

parallcl synthcsizcr (Sma Start Synthcsizcr, Chcm Spccd I-td., Switzelland) by rcfl xing the

rcaclrorr mrrrLrrcs lbr two lluurs at g0 jC. Srrc.s degrarlatinn studtcs wcrc pctlonn(.J by

maintaiDiDg r.rrliform degradation conditions using parallcl syrtllcsizcr (Snnrt Stalt Synthesizcr.

Clhcn Spccd Ltd., Switzerlafld) including acidio, ali(aline and reutral hydrolysis, oxidalion and

wct heati[g dcgradation. Oxidaiion at room temperature, photochernical and dr1. heating

dcgradation studies wele also carried out. After the raactiors were completed, all the solulioDs

wcrc prcscrvcd a1-80 'C till analysis.

,1 c i tl ic, .,1 I kaline a nd Ne ultu I I lldro lls is

lror acidic hydrolysis, 1N and 5N flCI werc uscd, lor alkalinc hydrol,vsis, 0.l N, lN aod 5N

NaOII were used wlile l'or neutral hydrolysis Milli ,Q waler was used. 3 rnl of each

conccntratiorl of acidic and alkaline solutions and Milti Q waler wcrc added into 3 nl, (l

nrg ml.) sto.,k solutiun. uf all thrcc scts

Wel IIeat Degrodtliqn

'l'o study wet heat degradation, 3 mL (l mg/ml) of stock solution of cach sct 4, sct 2 and set 5

was subiected to degradation.

164

Oxitlulion Reu:lion

Oxidation was cdrried out by adding I n1L of HrOz (35% v/v) in i mt. s1ocft sol(ioD ol cach sct.

All dre resultant solutions werc refluxed lor two hours at 80 "C in parallcl synthcsizer.

Dry lle Degradation

lJry ilcat degladaliorl was coDducted by takcll staodard Dexan'letl'lasoirc and Chlorarnphenicol

ancl hcatcd in oven at 90 "C ibr 4 hrs.

l'ltolo Degrulatiort

In order lo cvaluate photochcmical dcgradation of Dcxamethaso[e, Chloramphenicol and both in

cornbination, stock solution ofeach set was dircctly cxposcd to thc sunligl,t fbr thrce days fron 8

to 18 hrs at 30 + 2 "C. Separation was donc on TLC glass plates, prc coatcd with silica gcl 60 Ii

254 using chloroform : methanol (1.1:l v/v 2ld run). Avelage peak areas ofaclivc componenls

were aralyzeLl after triplicate analysis.

165

I)examcthasonc, I'rcdnisolonc Acctatc and Chloramphcnicol

McthaDolic stock solution (1 mg/ml) ofboth Dexamethasonc (set 4), prednisolonc acetac (set l)

and Chloramphcnicol (sct 2) was prcparcd, scparately aDd in combination (scts 3 and 5) to

pcfonn forced degradation studies in pal-allel syDthesizer (Smart Starl Synthosizcr. Chcm Spccd

Ltd., $witzerland) by rcfluxing the rcaction mixtures lor two hours a1 80 "C. Strcss dcgradation

stuclies were performed by maintaining uniforrn dcgradatio]1 condilions using parallel synthesizcr

(Snrart Stad Synthesizcr, Chem Spced Ltd., Switzerland) including acidic, alkalirc aurd noutral

irydlolysis, oxidation and wet heating degradation. Oxidaliofl at room lcmperature,

photochcmical and dry heating degradation studios wcrc also cafiied out. Aftcr the rc.totions

werc complctcd, allthe solutions were preserved at -80'C tiit analysis.

-1ci.lic, ,1ll@lin c tnd Neutrul llldtollsis

I;or aoidic hydrolysis, lN and 5N HCI wcre used, tbr alk. ine hydlolysis, 0.1N, lN and 5N

NaOII were used while for neutral hydrolysis Milli Q watel- was uscd. I ml- o1- cach

concentlatioll of acidic and alkaline solutioDs and Milli Q water were addcd into 3 mL (l

'nginl 1 sto. k solurlions ol cll rhre..sL.t..

\/el IIeal Degradalion

-lo study wct hoat degradalion, i m L ( I mg/ml) of stock solutio[ of cach sct I -5. was sLrbjcctcd

1o dcgradalion.

166

Oxidqtion Reaction

Oxidation was carricd out by addiDg 1 nll of Flror(j5,. !/v) in -'t urt- sl,,.k solulior ofcach

All the rosultant solutions were refluxed for two hour.s at 80 "C in parallcl synthesiz_cr.

Dr), Ileat Degrudstiott

Dry llcat dcgraclalion was condrcted by rakcn stanctard Dcxdncrhdso,rr lrlrd Chlorampheniool

aDd hcatcd il1 ovcn at 90 "C lor 4 h|s.

I'holo Degradqtion

ln or-der to cvaluatc photochemical degradatior of Dexamethasone, Chloramphenicol ancl both in

.onbina1iotl, stock solution ofeaoh set was directly cxposcd to the sunlighl for thrcc days fro[r g

to 18 l s at 1012 "C- Scpalation was done on TLC glass plates, pre-coatcd with silica gel 60 Ir

254 using chloroform: methanol (14:l \,/v) AvcrJge peak areas ofartivc lompollcnls worc

analyzed alicr lriplicate analysis-

3.4 'I'l,C I)cnsitomctry lnstrumcntation ,rnd pl.ramctcrs. Mcthod

Optimization and Validation

Plauar cl, romatograpl, y was pcrformed by spotting thc sample on TLC glass plate, pre-coatcd

willr silica gel 60F-254 (20 x 10 cm) with the aid of CAMAG microliter sample syringe using

CAMAG autoDratic'fLC l,inomat V applicator (Muntenz, Switzcr.land). A constant samplc

application rate ofo.l p[,/s was adoptcd and thc spaco betwocn the two lraDds was 6 mm. 15 ml,

of mobile phasc (chlorofonn: methanol, 14: I v/v 1br },rednisolone Acctalc and Chlor amphenicol,

161

chloroform: acetone, 8:2 v/v fbr l)examethasone and Chlommphenicol, chlorofom: methanol,

14:l v/v (2''d run) for Dexamethasone, Prednisolon€ Acetate and Chloramphen;col' in

combination)was used for linear ascending development and chromatogram was allowed to

moYotoadistalceof8cm,intwinlroughglasschamber(CAMAG).Forstressdegradation

studics, rnobile phase used were chloroform: methanol, 14:1 v/v for Prednisolone Acetate and

Chloramphenicol, chloroform: methanol, 14:1 v/v (2nd run) for Dexamethasone and

(lhloranphonicol, chlorofom: methanol, 14:1 v/v(2nd run)for Dexamethasone' Prednisolone

Acctalc and Chloramphenicol' in corrbination The chamber saturation time for mobile phase was

8 minutes at 25 l2 "C with relative humidity 42 1 5?n The dcvelopcd TLC plate wds dried with

thc help of air dryer for 4 min l)ensitometric scanning was performed on CAMAG Reprostar

scanncr Ill in the reflectance absorbance modc at multiwavelength ('tnu' 238'5 nm for

Dcxamerhasone;,trn**, 243 nnr for Prednisolone Acctate and,t.".,278 nm for chloramphenicol)

by utilizing deuterium lamP as the solrrce ofradiation Quantitative evaluation was perrormed via

pcak areas by Wincats software (version 1 2 3) Densitometric scanning parameters were as

lbllows: bandwidth: l0 mm, slit width: 0.45 mm' slit length: 5 mm' scanning speed: 10 mm/s'

l hc devcloped method was vatidated as per the requirements ofthe ICH guidelines'

l,inearity

Linearity was evaluated by determining six

r'0n0 ng sfot. Peak area rnd concenlration

equatiotl to calculate thc regression data and

standard working solutions at a concentration 200-

was subje(led lo lhe least square linear regression

correlation coeffi cients.

158

Precision and AccuracY

lntra day al1d inter-day precisions were determined with the standards and degraded rcaction

mixtures. For method repeatability, assay at three d;fferent concentration levels (200' 400 and

800 ng) was repealedly performed six times on the same day (intra-day) For reproducibility'

same samples at three concentration levels (200, 400 and 800 ng) were analyzed at different days

(inlcFday) anLl results were slatistically evaluated in terms ofTo R S D'

Limit of Dctcction and Limit of Quantilicntion

ln order to calculate Sn',1 ratio for

rcspcctively where 5 is the residual

Robustness and SPccificitY

LOD and LOQ, the fomulae used were 3 3 6/5 and l0 6/5'

crror and S stands for slope ofcalibration curve'

ln order to check the robustncss, following parameters were deliberately changed within the

rangc of t 5% at three difierent concentration levels (200' 400 and 800 ng); amount of mobile

phasc, mobile phase composition, time from spotting to chromatography' time from

chromatography to scanning and chamber saturation time'

RccoYcry Studies

lor recovcry studies, pre analyzcd pharmaceutical drugs containing active components

individually and in combination were spiked with extra 25, 50 and 757o of analyzed drug' The

specillcity ofthc proposed method was analyzed by overlapping the densitogram ofthe standard

and samples and comparing it at peak start and peak end positions'

169

In order 10 develop validated TLC densitometry mcthod Ior determination of ChloramPhenicol'

Dexarrethasone and Prednisolone Acetate,individually and in combination, 6 pL of each sample

(Pl, P2, Pl, P4 and P5; Dl, D2 D 3 and D4; and Cl, C2. C3 and C4) and PCl' PC2' DCI'

DC2and expired products EC I , EPC 1 and EDC I were applied on TLC plate for chromatographic

)ror soess dcsradation stud;es, 1 pL (s00 ngApot) of lN HCl, 0 5 pL (250 nelspot) of 5N HCI' 4

UI- (2000 ng/spot) of 0 lN, lN and 5N NaOH treated solutions) 4 pL (2000 n8/spot) of neutral

hydrolysis, 6 pI- (6000 ng/spot) from wet heat degradation mixture and 4 pL (3000 ng/spot)

ftom oxidation mixture were applied on TLC plate in iriPlicate for chromatographic analvsis'

For dry hcat strcss studies, I mg olcach lrcated standard was dissolved in 1 mL ofmcthanol and

4 rrl. (4000 nB/spot) of resultant solutions were applied on TLC plat€ in triplicate for

ohromatographic analysis.6 prL (6000 ng/spot) ofeach sun Iight treated solution Dexamethasone'

I'rednisolone Acetalc, Chloramphenicol and Dexamethasone and Chloramphenicol' and

prednisolone AceLate and chloramphenicol in combination were applied on TLC plate in

triplicatc lor chromatographic analysis.

110

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Liu llL. Yang lt, Li F, Ma YJ: Detennination ofseven antibiotics i!1 cosmetics bv ulrra

performance liquid cht-omatography.J r,v'l' n Hetthh2oj9 ' 26 :453-454'

6l LiLr Ht,, t,i F. Yang R, Wang LH, Ma YJI Determination of common antibiotics iud

metronidazole in cosmetics by ultra performance liquid chromatographytandem mass

spectrometry- Crire.tu .l Chromatogr 2009,21 :50-53'

Sethi PD: HPTLC high performance thin layer chromatography quantitative analysis of

phannaceutical formulations. CBS: Ncw Dehli, 1996'

ICII Q2 (Rl) Validation ofanalyticat procedures: text and methodoloev ln: procecdings

ofthe hletnational Conlcrence on Ilannonization, 1995'

6,1.

65.

177

66. tCH QIA 0f2) Stability tcsting of ncw drug substances and products. lni procecdings of

the lnternational Confcrcncc on Ilarmonization. 2003.

tl8

5.0 PUBLICATIONS

119

1, Author of book titled "Microbial Transformatior of Steroid and Telpene" published by

LAt'LAMBEI{TAcademicPublishingGmbH&CoKG'ISBN:978-3-8465-1222-7

2. Sycd G. Musharraf, Urooj.latima and Rahat Sultana"'Sfiess degradation studies

an<J development of stability-indicating TLC-densitometry methotl for determination of

Prednisolone Acetate and Chloramphenicol in their individual and combined

plrallnaceutical fol1n u|aliol'\s..,Chemi try Cenlral Journal 2o]r2, 6i 7, Pnhlished: 22

January 20 1 2, doi: 1 0.1 1861 17 52'153X-6-7

-1. Sadia Sultan; M lqbal Choudhary; Shamsun Nahar Khan; Urooj Fatima; Muhammad

Atif; ltahat Azhrr AIi; Atta-Ur- Rahman;M Qaiser Falmi,..Fungal transformation of

cedryl acetate and o-glucosiclase inhibition assay' quantum mechanical calculations and

nrolccular doching studics of its metabolites "ElroPean journal of medicinctl

chemistrY 2013 62:7 64-10 '

4. Sycd G. Musharraf, Urooj Fatima and Rahat Azhar Ali' "Validatcd I Iltra-liast

llPl,C method 1'or sirnultancous determination ofPrcdnisolone Acetate and

Chiorampheniool in pharmaceutical prod ncts " ManuscriPt submitted

180