Sensitive and Robust Methods for Simultaneous Determination of BeclomethasoneDipropionate and Formoterol Fumarate Dihydrate in Rotacaps

Vijaykumar K. Parmar*, Hetvi N. Patel and Bhavin K. Patel

Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, CHARUSAT Campus, Ta. Petlad, Dist. Anand,

Changa 388421, Gujarat, India

*Author to whom correspondence should be addressed. E-mail: vijayparmar.ph@charusat.ac.in; vijaykparmar@gmail.com

Received 31 May 2013; revised 8 December 2013

Fixed dose combination containing beclomethasone dipropionate(BDP) and formoterol fumarate dihydrate (FFD) is used in the treat-ment of asthma in form of dry powder inhaler. Two methods aredescribed for the simultaneous determination of BDP and FFD incommercial rotacap formulation. The first method was based onHPTLC separation of the two drugs followed by densitometric mea-surements of their spots at 220 nm. The separation was carried outon Merck HPTLC aluminum sheets precoated with silica gel 60F254using hexane:ethyl acetate:methanol:formic acid (2.0:2.5:2.0:0.2,v/v/v/v) as mobile phase. The linearity was found to be in the rangeof 2.4–8.4 mg/spot and 80–280 ng/spot for BDP and FFD, respect-ively. The second method was based on HPLC separation of the twodrugs on the reversed phase Enable HPLC Analytical C18 G 120A(250 3 4.6 mm, 5 mm) column at ambient temperature using amobile phase consisting of methanol:acetonitrile:phosphate bufferadjusted to pH 3.6 using orthophosphoric acid (65:25:10, v/v/v).Quantitation was achieved with UV detection at 220 nm based onpeak area with linear calibration curves at concentration ranges of10–200 and 0.3–6.0 mg/mL for BDP and FFD, respectively. Bothmethods were validated in terms of precision, robustness, recoveryand limits of detection and quantitation. The robustness of bothmethods was assessed using experimental design and results wereanalyzed by statistical and graphical approaches. Rotacaps formula-tion containing BDP (200/400 mg) and FFD (6 mg) were successfullyquantified using the proposed methods. The proposed methods canbe used as sensitive, precise, accurate and robust methods for quan-tification of BDP and FFD in Rotacaps.

Introduction

Asthma is the commonest chronic disease in children in eco-

nomically developed countries and is also common in adults (1).

It is increasing in prevalence and severity. Asthma is associated

with airway inflammation, airway hyperreactivity and acute bron-

chial constriction (2). The drugs used most commonly in the

treatment of asthma, b2 adrenergic receptor agonists and gluco-

corticoids, have potentially serious side effects when delivered

systemically (3). The aerosol delivery of these drugs produces

a high local concentration in the lungs with a low systemic

delivery, thereby significantly minimizing systemic side effects.

Aerosol delivery in the forms of nasal sprays, metered-dose

inhalers (MDI), dry powder inhalers (DPI) and nebulizers, are

commonly used for treatment of asthma and chronic obstructive

pulmonary disease (COPD) (4). The most of the aerosol

products for asthma have been launched in combination like

b2-adrenoreceptor with corticosteroids for prolonged duration

of action, more effectiveness and quick relief. A significant

synergistic therapeutic effect can be obtained in the treatment of

asthma by using a fixed dose combinations containing formoterol

fumarate dihydrate (FFD) (6 mg) and beclomethasone dipropio-

nate (BDP) (200/400 mg) in form of dry powder inhaler (5).

BDP, 9a-chloro-11b-hydroxy-16b-methyl-3,20-dioxopregna-

1,4-diene-17,21-diyldipropionate (Figure 1a), is a glucocorticoid

steroid (6). The anti-inflammatory effects of BDP in asthma

include modulation of cytokine and chemokine production;

inhibition of eicosanoid synthesis; marked inhibition of accumu-

lation of basophils, eosinophils and other leukocytes in lung

tissue; and decreased vascular permeability (2). The HPLC

methods are reported for assay of BDP in bulk in Indian

Pharmacopoeia (7), British Pharmacopoeia (8) and European

Pharmacopoeia (9). BDP inhaler formulation is official in Indian

Pharmacopoeia mentioning HPLC method for the assay of BDP.

Dhudashia et al. reported stability indicating HPLC method for

estimation of BDP from cream (10). The UPLC (11), HPLC (12)

and spectrophotometric (13) methods were reported for simul-

taneous estimation of BDP and Salbutamol in bulk and Rotacaps.

Also determination of BDP in biological fluids by liquid chroma-

tography–mass spectrometry (14) and HPLC (15) methods has

been reported.

FFD, (RS)-20-hydroxy-50-[(RS)-1-hydroxy-2 [[(RS)p-methoxy-

a-methylphenethyl]amino]ethyl] formanilide (Figure 1B), is a

long-acting b2-agonist (6). It is used in the management of

asthma and/or COPD (4). Inhaled formoterol works like other

b2-agonists, causing bronchodilatation by relaxing the smooth

muscle in the airway so as to treat the exacerbation of asthma

(2). Potentiometric (7–9), spectrophotometric (16, 17) and

HPLC (18, 19) methods are reported in the literature for deter-

mination of FFD in bulk and pharmaceutical formulation. Several

methods are reported for simultaneous determination of compo-

nents of fixed dose combination product containing FFD and

glucocorticoids such as fluticasone, budenoside and ciclesonide

in the form of pulmonary product (20–23). Determination of

formoterol in urine by liquid chromatography–tandem mass

spectrometry is also reported (24).

From the literature survey, it was found that there is no known

method available for the simultaneous determination of these

two drugs in the commercially available formulation. Therefore,

it was thought of interest to develop analytical methods for

the simultaneous determination of BDP and FFD in Rotacaps.

Two methods, HPTLC and HPLC, were developed and validated

for determination of both drugs from marketed formulation.

# The Author [2014]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Journal of Chromatographic Science 2014;1–12

doi:10.1093/chromsci/bmt208 Article

Journal of Chromatographic Science Advance Access published February 9, 2014

An experimental design approach was used for determination of

robustness of the proposed methods as a part of an overall

method validation strategy. The robustness testing is generally

performed by evaluating the effects of individual factors by

varying one variable at a time. The use of experimental design

helps to predict the possible interactions between the factors

with limited number of experiments. The design of experiment

(DOE) approach supports ICH guidelines of applying quality by

design (QbD) to predict the parameters negatively affecting the

quality of product (25). The proposed methods are found to be

sensitive and suitable for routine quality control of formulation

containing BDP and FFD.

Experimental

Instrumentation

The HPTLC system (Camag Sonnenmattstr, Mutenz, Switzerland)

consisting of a Linomat V semi-automatic spotting device, a glass

twin-trough TLC chamber (20 � 10 cm), a TLC scanner-IV, a

data station with winCATS (V 1.4.7) software and an HPTLC

syringe (100 mL capacity; Hamilton Company, NV, USA) was

used for thin layer chromatographic studies.

The HPLC system (Shimadzu Corporation, Analytical Instruments

Division, Kyoto, Japan) consisting of a Rheodyne syringe loading

sample injector (20 mL), an LC-AT20 solvent delivery module,

SPD M20A PDA detector, CTO-10AS column oven, LC solution

work station was used for LC separation. The chromatographic

separation was accomplished on an Enable HPLC Analytical C18

G 120 A (250 � 4.6 mm, 5 mm) column protected with a guard

column of the same stationary phase.

Chemicals

BDP (Batch no. BMD 0160811) was procured as gift sample from

Tripda Biotech Private Limited, Ahmedabad, India. FFD (Batch

no. FF-0020610) was supplied as gratis sample from Cadila

Healthcare Ltd., Ahmedabad, India. Drug substances were used

without further purification and certified to contain 99.89%

(w/w) purity for BDP and 99.20% (w/w) purity for FFD on dried

basis. Acetonitrile, ethyl acetate, formic acid, hexane, methanol,

orthophosphoric acid and potassium dihydrogen orthophosphate

were procured from Loba Chemicals, Mumbai, India. The HPLC

grade chemicals and reagents were used for HPLC method. Glass

distillation assembly from Durga scientific, Vadodara was used to

prepare triple distilled water. Marketed formulation containing

BDP and FFD were procured from local market.

Chromatographic conditions

HPTLC method

Separation was performed on precoated silica gel aluminium

plate 60 F-254 (20 � 10 cm) with 250 mm thickness; E. Merck,

Darmstadt, Germany, supplied by Anchrom Technologists

(Mumbai). The TLC plate was prewashed with methanol and

dried in an oven at 1108C for 5 min. Samples were spotted on

the TLC plate in the form of band leaving 10 mm from the

bottom edge using Linomat V semi-automatic spotter and ana-

lyzed using following parameters: bandwidth, 6 mm; track dis-

tance, 10 mm; spraying rate, 150 nL/s; volume of mobile phase,

6.5 mL; temperature, 27+18C; chamber saturation time, 10 min;

migration distance, 70 mm; slit dimension, 4.00 � 0.30 mm;

scanning speed, 20 mm/s; detection wavelength, 220 nm.

Mobile phase consisted of hexane:ethyl acetate:methanol:formic

acid (2.0:2.5:2.0:0.2, v/v/v/v).

HPLC method

The mobile phase consisted of methanol:acetonitrile:phosphate

buffer adjusted to pH 3.6 with orthophosphoric acid (65:25:10,

v/v/v). Samples were analyzed using the following parameters:

flow rate, 1.0 mL/min; injection volume, 20 mL; run time, 6 min;

temperature, 27+28C; detection wavelength, 220 nm.

Preparation of stock solutions

Accurately weighed 100 mg of BDP was transferred to 100 mL

volumetric flask, dissolved and diluted up to the mark with

methanol to get BDP stock solution containing 1 mg/mL of BDP.

Accurately weighed 100 mg of FFD was transferred to 100 mL

volumetric flask, dissolved and diluted up to the mark with

methanol to get FFD stock solution containing 1 mg/mL of FFD.

Preparation of calibration curve

For HPTLC method

The combined working standard solution of BDP and FFD was

prepared by diluting the stock solutions with methanol to

prepare mixture of 300 mg/mL of BDP and 10 mg/mL of FFD.

Aliquots of 8, 12, 16, 20, 24 and 28 mL of working standard solu-

tion (corresponding to 2.4, 3.6, 4.8, 6, 7.2, 8.4 mg/spot and 80,

120, 160, 200, 240, 280 ng/spot for BDP and FFD, respectively)

were spotted on a TLC plate and analyzed. Calibration curve was

Figure 1. Structure of (A) BDP and (B) FFD.

2 Parmar et al.

prepared by plotting peak area of BDP and FFD against their re-

spective concentration.

For HPLC method

The working standard solutions of BDP and FFD were prepared

by diluting stock solutions with mobile phase to prepare

400 mg/mL of BDP and 20 mg/mL of FFD solutions. Aliquots

(0.25, 0.50, 1.0, 2.0, 4.0 and 5.0 mL) from working standard solu-

tion of BDP (400 mg/mL) and aliquots (0.15, 0.3, 0.6, 1.2, 2.4 and

3.6 mL) from working standard solution of FFD (20 mg/mL)

were diluted up to 10 mL with mobile phase to prepare mixture

of calibration standard solutions corresponding to 10, 20, 40, 80,

160 and 200 mg/mL of BDP and 0.3, 0.6, 1.2, 2.4, 4.8, 6.0 mg/mL

of FFD. The calibration standard solutions were analyzed by the

proposed procedure. Calibration curve was prepared by plotting

peak area of BDP and FFD against their respective concentration.

Sample preparation

For assay

To determine the content of BDP and FFD simultaneously in

marketed formulations (formulation A: labeled claim: 200 mg

BDP and 6 mg FFD in each capsule; Formulation B: labeled claim:

400 mg BDP and 6 mg FFD in each capsule), the 20 capsules

were weighed and emptied. The capsule powder was transferred

to 25 mL volumetric flask. Five milliliters of methanol was added.

The capsule shells were rinsed twice with methanol (5 mL),

dried and weighed. The methanolic extract was transferred to

volumetric flask containing capsule powder. The solution was

sonicated for 25 min, diluted up to the mark with methanol and

filtered using 0.45 mm filters (Millipore, Milford, MA, USA) to

get the sample solution containing 160 mg/mL of BDP and

4.8 mg/mL of FFD for formulation A and 320 mg/mL of BDP and

4.8 mg/mL of FFD for formulation B.

For content uniformity

Ten capsules were taken for content uniformity test. Each capsule

was weighed and emptied. The content of each capsule was trans-

ferred to a series of 10 mL volumetric flasks. The capsule shells

were rinsed with methanol and the rinsing solution was trans-

ferred to volumetric flask. The volume was made up to the mark

with methanol. The mixtures were sonicated for 25 min. The

resulting solution was filtered using 0.45mm filters (Millipore,

Milford, MA, USA) to get the sample solution containing 20

and 0.6 mg/mL of BDP and FFD (for formulation A) and 40 and

0.6 mg/mL of BDP and FFD (for formulation B), respectively.

Validation of the proposed methods

Both the methods were validated in compliance with ICH guide-

lines (26, 27). The following parameters were validated.

Specificity

The specificity of the methods was ascertained by analyzing

standard drug and sample solutions.

HPTLC method

The spot for BDP and FFD in sample solution prepared from

marketed formulation was confirmed by comparing the Rf and

absorbance/reflectance spectrum with that of standard BDP and

FFD. The peak purity of BDP and FFD was assessed by correlating

the spectra at three different levels, i.e., peak start (S), peak apex

(M) and peak end (E) positions of the spot.

HPLC method

The chromatograms of standard and sample solutions containing

BDP and FFD were compared to determine the specificity of the

HPLC method. The retention time of BDP and FFD was found to

check selectivity of the method. The PDA (photo diode array)

spectrum of drug peaks obtained from sample and standard solu-

tions were compared. Peak purity analysis was performed using

LC solution software. The specificity was further determined by

the complete separation of BDP and FFD along with other para-

meters like retention time (Rt), capacity factor (k), tailing or

asymmetrical factor (T), etc.

Linearity

Standard stock solution of the drug was diluted to prepare lin-

earity standard solutions containing BDP and FFD in the concen-

tration range of 2.4–8.4 mg/spot, 80–280 ng/spot and 10–

200 mg/mL, 0.3–6.0 mg/mL for HPTLC and HPLC method, re-

spectively. Six sets of such solutions were prepared. Each set

was analyzed to plot a calibration curve. Standard deviation (SD),

slope, intercept and coefficient of determination (r2) of the cali-

bration curves were calculated to ascertain linearity of the

method.

Precision

The precision is measure of either the degree of reproducibility

or repeatability of analytical method. It is indication of random

error. The precision of analytical method is expressed as a SD,

relative standard error or coefficient of variance of series of

measurement.

HPTLC method

Repeatability of measurement of peak area was carried out by

repeated scan of the same spot (4.8 mg/spot of BDP and 160 ng/spot of FFD) seven times without changing the plate position.

The % RSD for peak area was calculated. Repeatability of sample

application is based on seven-time application of combined

standard solution. The % RSD for peak area was computed.

Variations of results within same day (intraday precision) and

among days (interday precision) are called as reproducibility.

The intraday precision (% RSD) was determined by analyzing

standard solution of BDP and FFD for three times on the same

day. The interday precision (% RSD) was determined by analyz-

ing standard solution of BDP and FFD for 5 days. The intra- and

interday variation for determination of BDP and FFD was carried

out at three different concentration levels 3.6, 4.8, 6.0 mg/spotof BDP and 120, 160, 200 ng/spot of FFD.

HPLC method

System suitability tests are used to verify that resolution and re-

producibility of chromatography. It was performed by injecting

20 mL of standard solution, containing 200 mg/mL of BDP and

6 mg/mL of FFD, six times. Resolution (R), column efficiency

(N), tailing factor (T) and precision of injection repeatability

were analyzed.

Sensitive and Robust Methods for BDP and FFD 3

The intraday precision (% RSD) was determined by analyzing

standard solution of BDP and FFD for three times on the same

day. The interday precision (% RSD) was determined by analyz-

ing standard solution of BDP and FFD for 5 days. The intra- and

interday variation for determination of BDP and FFD was carried

out at three different concentration levels 40, 80, 160 mg/mL of

BDP and 1.2, 2.4, 4.8 mg/mL of FFD.

Recovery studies

The accuracy was determined by the standard addition method.

To a fixed amount of pre-analyzed sample of BDP and FFD, in-

creasing amount of standard BDP and FFD solution was added.

HPTLC method

Accuracy of an analysis is determined by calculating systemic

error involved. The recovery studies were carried out by apply-

ing the method to drug sample to which known amount of BDP

and FFD corresponding to 80, 100 and 120% of label claim had

been added. At each level of the amount, three determinations

were performed and the results obtained were compared with

expected results.

HPLC method

Recovery study was performed by addition of known amounts of

standard drugs to pre-analyzed commercial pharmaceutical product

sample at three different concentration levels of drug (50, 100 and

150%). Along with sample solution of BDP (40 mg/mL) and FFD

(1.2 mg/mL) working standard solution of BDP and FFD at each

level were added to prepare mixtures of BDP (6080, 100 mg/mL)

and FFD (1.8, 2.4, 3.0 mg/mL). The experiment was repeated three

times.

Robustness

Robustness testing was performed by experimental design ap-

proach (28–31). Plackett–Burman (PB) designs for testing of

seven factors are the most commonly used designs for robust-

ness testing of chromatographic methods. The proposed HPTLC

method was tested for robustness using PB design with eight

experiments. Seven HPTLC conditions were screened: (A)

change in amount of hexane in mobile phase composition, (B)

change in amount of ethyl acetate in mobile phase composition,

(C) change in amount of methanol in mobile phase composition,

(D) change in saturation time, (E) change in detection wave-

length, (F) change in bandwidth and (G) change in solvent run

distance (Table Ia). Robustness of HPLC method was determined

using two-level 24–1 fractional factorial design. The generator

for this design, that has resolution IV, was D ¼ ABC. The

procedure-related factors examined were: (P) change in mobile

phase composition, (Q) change in pH of mobile phase, (R)

change in flow rate and (S) change in detection wavelength

(Table Ib).

The DOEþþ (Reliasoft Corporation, AZ, USA; ver 1.0.7) and

Design of Experiment (Stat Ease, MN, USA; ver 8.0.1) software

were used to set up the experimental designs for HPTLC and

HPLC methods, respectively. The % recoveries and Rf values for

BDP and FFD were observed at each experiment designed for

the HPTLC method, whereas % recoveries and resolution

between FFD and BDP were observed for the HPLC method. The

experiment was repeated three times. The experiments were

executed in random order. The significance of the factor effects

was determined statistically, using error estimates in the calcula-

tion of critical effects, and graphically, by means of half-normal

plots and Pareto charts.

Limit of detection and limit of quantitation

The detection limit of an individual analytical procedure is the

lowest amount of analyte in a sample that can be detected but

not necessarily quantitated as an exact value. The quantitation

limit of an individual analytical procedure is the lowest amount

of analyte in a sample that can be quantitatively determined

with suitable precision and accuracy. The quantitation limit is a

parameter of quantitative assays for low levels of compounds in

sample matrices and is used particularly for the determination

of impurities and/or degradation products. The limit of detec-

tion (LOD) and limit of quantitation (LOQ) were separately

determined at a signal-to-noise (S/N) ratio of 3 and 10. LOD

and LOQ were experimentally verified by diluting known

concentrations of BDP and FFD until the average responses

were �3 or 10 times the SD of the responses for six replicate

determinations.

Table IFactors and Their Levels for Robustness Testing of HPTLC and HPLC Methods

Factors Levels

(2) Nominal (0) (þ)

(a) HPTLC method(A) Change in amount of hexane in mobile phase composition (mL) 1.8 2.0 2.2(B) Change in amount of ethyl acetate in mobile phase composition (mL) 2.25 2.50 2.75(C) Change in amount of methanol in mobile phase composition (mL) 1.8 2.0 2.2(D) Change in saturation time (min) 9 10 11(E) Change in detection wavelength (nm) 219 220 221(F) Change in bend width (mm) 4 6 8(G) Change in solvent run distance (cm) 6.5 7.0 7.5

(b) HPLC method(P) Change in amount of methanol in mobile phase composition (mL) 62 65 68(Q) Change in pH of mobile phase 3.5 3.6 3.7(R) Change in detection wavelength (nm) 219 220 221(S) Change in Flow rate (mL/min) 0.95 1.00 1.05

4 Parmar et al.

Analysis of marketed formulations

Assay

HPTLC method. Sample solution (25 mL) was spotted on

TLC plate in triplicate. The plate was developed and analyzed. The

peak area of the spots were measured for BDP and FFD and their

concentrations in the samples were determined using multilevel

calibration curves developed on the same plate under the same

conditions using linear regression equation.

HPLC method. From formulation A sample solution, a 20-mL

volume of solution (4.8 and 160 mg/mL of FFD and BDP,

respectively) was chromatographed in HPLC system, three times.

The peak area of the BDP and FFD was measured at 220 nm, and

their concentrations in the samples were determined using linear

regression equation.

From formulation B sample solution, 2 mL was pipette out

to 10 mL volumetric flask and dilute up to the mark with

mobile phase to get concentration 32 and 0.96 mg/mL of BDP and

FFD, respectively. A 20-mL volume of the resulting solution was

injected in HPLC system, three times. The peak area of the BDP

and FFD was measured at 220 nm and their concentrations in the

samples were determined using linear regression equation.

Content uniformity

A 20 mL volume of the sample solution for content uniformity

was injected in HPLC system, three times, and analyzed. The

peak area of the BDP and FFD was measured at 220 nm and their

concentrations in the samples were determined using linear

regression equation. The content uniformity for the marketed

formulations was tested as per USP guidelines (32).

Results

Chromatographic separation

HPTLC method

Various solvent systems composed of toluene, methanol, ethyl

acetate, hexane or mixture thereof were tried for optimization

of mobile phase for HPTLC separation of BDP and FFD. But

the best resolution and symmetrical peak shapes were achieved

using mobile phase system consisting of hexane:ethyl acetate:

methanol:formic acid (2.0:2.5:2.0:0.2 v/v/v/v). The Rf values

were found to be 0.36 and 0.68 for FFD and BDP, respectively.

The comparison of densitograms of standard drug solution and

sample solution from formulation showed identical Rf values, i.e.,

0.68 for BDP and 0.36 for FFD (Figure 2). Comparison of the

spectra scanned at peak start (S), peak apex (M) and peak end (E)

positions of individual spots of BDP and FFD obtained from

sample solution showed a high degree of correlation (.0.99),

confirmed the purity of the corresponding spots (Supplementary

Figure 1). The spectrum of individual drug was also correlated

with spectrum of standard BDP and FFD. The correlation obtained

was 0.9998 and 0.9991 for BDP and FFD, respectively, confirmed

the identity of the spots.

HPLC method

The mobile phase system consisting of methanol:acetonitrile:

water (65:25:10, v/v/v) adjusted to pH 3.6 showed good

separation, good peak shape and repeatable result. The elution

of both drugs from HPLC column was achieved at 1.69 min for

FFD and 4.78 min for BDP (Figure 3). The results of the system

suitability test (Table II) shows that the optimized chromato-

graphic conditions are adequate for simultaneous determin-

ation. The resolution between BDP and FFD peaks was found to

be 17.45. Chromatograms of standard drug solution and sample

solution were compared and it showed identical retention

times for both drugs (Figure 3). The peak purity indices for

both drugs from standard and sample solution were found to

be .0.999.

Method validation

HPTLC method

Representative calibration curve of BDP and FFD was obtained

by plotting the peak area of BDP and FFD against concentration

over the range of 2.4–8.4 mg/spot and 80–280 ng/spot, respect-ively. They were found to be linear over above-mentioned range

with correlation coefficients of 0.9979+0.001 for BDP and

0.9978+0.0006 for FFD. The linear regression equations were

y ¼ 1907.8x þ 2822.2 and y ¼ 13.21x 2 73.794 for BDP and

FFD, respectively. The repeatability of measurement of peak area

and sample application were expressed in terms of % RSD and

were found to be 0.88, 0.82 and 1.47, 1.33 for BDP and FFD,

respectively. The % RSD for intraday precision (Table III) was

found to be 0.17–0.85 and 0.17–0.48 for BDP and FFD, respect-

ively. The % RSD for interday precision (Table III) was found

to be 0.32–1.62 and 0.72–1.19 for BDP and FFD, respectively.

The proposed HPTLC method when used for recovery studies

for BDP and FFD from pharmaceutical formulation after spiking

with additional standard drug afforded recovery between

98–102% and mean recoveries for BDP and FFD from the

marketed formulation are listed in Table IV. The LOQs and LODs

were found to be 0.44 mg/spot, 0.14 ng/spot for BDP and

10.94 ng/spot, 3.61 ng/spot for FFD, respectively. The total

eight experimental plans of PB design for robustness testing and

the corresponding responses are summarized in Table V. The

margin of errors (ME) or the critical effects were calculated for

each response using algorithm of Dong (30). The values for ME

are shown in Table V. Pareto charts evaluation of robustness data

are depicted in Figure 4.

The solution stability and spot stability studies were per-

formed for the HPTLC method. Solutions of BDP (160 ng/mL)and FFD (4.8 ng/mL) were prepared from sample solution and

stored at room temperature for 0.5, 1.0, 2.0, 4.0, 8.0 and 24 h,

respectively. They were then applied on the TLC plate, after

development of the densitogram was evaluated for additional

spots if any. There was no indication of compound instability in

the sample solution. The time the sample is left to stand on the

solvent prior to chromatographic development can influence

the stability of separated spots and are required to be investi-

gated for validation. Two-dimensional chromatography using

same solvent system was used to find out any decomposition

occurring during spotting and development. In case, if decom-

position occurs during development, peak(s) of decomposition

product(s) shall be obtained for the analyte both in the first and

second direction of the run. No decomposition was observed

during spotting and development.

Sensitive and Robust Methods for BDP and FFD 5

HPLC method

BDP and FFD showed good correlation coefficient in the con-

centration range of 10–200 mg/mL (r ¼ 0.9980+0.001) and

0.3–6.0 mg/mL (r ¼ 0.9976+0.0005) for the HPLC method, re-

spectively. The linear regression equations were y ¼ 13,329x þ

48,850 and y ¼ 54,558x þ 7,030 for BDP and FFD, respectively.

The SD for slope and intercept calculated was 132.07 and

3742.65 for BDP and 397.94 and 806.68 for FFD, respectively.

Intraday precision (%RSD, n ¼ 3) was found to be 0.21–0.88 and

0.16–0.95 for BDP and FFD, respectively. Interday precision

Figure 2. Densitogram of (A) standard BDP (3.6 mg/spot, Rf: 0.68+ 0.02) and FFD (120 ng/spot, Rf: 0.36+ 0.02), (B) sample BDP (3.6 mg/spot, Rf: 0.68+ 0.02) and FFD(120 ng/spot, Rf: 0.36+ 0.02) measured at 220 nm, mobile phase hexane–ethyl acetate–methanol–formic acid (2.0:2.5:2.0:0.2, v/v/v/v).

6 Parmar et al.

(%RSD, n ¼ 3) was found to be 1.10–1.24 and 0.84–1.25 for

BDP and FFD, respectively. The intra- and interday precision

result is depicted in Table III. The accuracy of the method was

evaluated by recoveries of the added sample. The results of re-

covery studies for the HPLC method are presented in Table IVb.

The LOQs were found to be 5.06 and 0.14 mg/mL for BDP and

FFD, respectively, and the LODs were estimated to be 1.67 and

0.05 mg/mL for BDP and FFD, respectively. Standard solution of

BDP (80 mg/mL) and FFD (2.4 mg/mL) were prepared from

sample solution and stored at room temperature for 3 days. They

were then injected into the HPLC system and no additional peak

was found in the chromatogram indicating the stability of BDP

and FFD in the sample solution.

The statistical analysis of the robustness testing data was done

by Design Expert software. The significance of the effects was

evaluated by the statistical interpretation method (Algorithm of

Dong) and graphical methods, namely a half-normal probability

plot of the residuals and Pareto chart of the standardized

effects. The half-normal plots showing effect of factor change on

responses are depicted in Figure 5. The factor effects were

calculated for each response and presented in Table VI.

Analysis of the marketed formulation

The proposed methods were applied for assay of BDP and FFD

from marketed formulations. The spots at Rf 0.68 (for BDP) and

Rf 0.36 (for FFD) were observed in the densitogram of the drug

samples extracted from capsules (Figure 2B). There was no

interference from the excipients commonly present in the cap-

sules. The HPLC chromatogram of the drug samples extracted

from capsule showed peaks at retention time of 4.78 min (for

BDP) and 1.68 min (for FFD) (Figure 3B). The HPLC method was

further applied for content uniformity testing of marketed

formulation. The steps of the test were adopted according to the

USP procedure (32). The acceptance value (AV) was calculated

for each of the Rotacaps and was found to be smaller than the

maximum allowed acceptance value (LI).

Comparison between HPLC and HPTLC methods

Sample solutions were analyzed simultaneously by HPTLC and

HPLC methods. Each sample was analyzed in triplicate. The results

of both methods were compared by paired t-test. The data were

treated as paired data. The statistical data are shown in Table VII.

Discussion

By reviewing the literature in hand, it was found that no chroma-

tographic methods were published for the simultaneous deter-

mination of binary mixture of BDP and FFD. Therefore, the aim

of this work was to develop and validate chromatographic

methods for simultaneous determination of the cited drugs.

Optimization of procedure

HPTLC method

Initially, toluene and methanol in the ratio of 5:5 (v/v) were tried

for separation of both drugs simultaneously. The spots were not

developed properly and dragging was observed. Then toluene and

methanol in the ratio of 3:7 (v/v) was tried. The developed spots

were diffused and Rf was near to solvent front. Then the reverse

ratio of the same mobile phase was tried. The distance travelled by

developed spots was less and dragging was observed. To the

above mobile phase, ethyl acetate was added with different ratios

but the spot of BDP was diffused to solvent run, then ethyl acetate

was replaced with ethanol but the developed spots lack compact-

ness and were observed to be less persistent. Also the Rf values of

FFD and BDP were not satisfactory because of less resolution

between them. Then toluene was replaced by hexane and ethanol

was again replaced by ethyl acetate. Different ratios of hexane,

ethyl acetate and methanol were tried and finally 2.0:2.5:2.0 (v/v/v) ratio of hexane, ethyl acetate and methanol was optimized. For

improvement in peak shape of both the drugs, 0.2 mL of formic

acid was added to hexane, ethyl acetate and methanol in the ratio

of 2:2.5:2:0.2 (v/v/v/v).The densitometric scanning at 220 nm, the iso-absorptive point

determined from overlain absorbance/reflectance spectra of FFD

and BDP, resulted in increased sensitivity of the method.

Well-defined spots were obtained when plate was activated at

1108C for 15 min and the chamber was saturated with the mobile

phase for 10 min at room temperature. It was observed that drying

of TLC plate after spotting and pre-saturation of TLC chamber

with the mobile phase for 10 min ensured good reproducibility of

Rf value. The proposed HPTLCmethod has the advantage of allow-

ing determination of several samples at the same time.

Figure 3. HPLC chromatograms of (A) sample solution of BDP (160 mg/mL, Rt:4.656 min) and FFD (4.8 mg/mL, Rt: 1.664 min), (B) working standard solution of BDP(160 mg/mL, Rt: 4.655 min) and FFD (4.8 mg/mL, Rt: 1.663 min) at 220 nm; mobilephase:methanol:acetonitrile: 25 mM phosphate buffer, pH 3.6, adjusted withorthophosphoric acid in ratio of 65:25:10 (v/v/v).

Sensitive and Robust Methods for BDP and FFD 7

HPLC method

The main problem in the development of a method for the con-

temporary analysis of BDP and FFD was to find a suitable combin-

ation of mobile phases to separate the components. Preliminary

isocratic studies on a reverse-phase C18 column with different

mobile phase combinations were tried. According to the litera-

ture review, initially, acetonitrile and water were tried in the

ratio of 80:20 (v/v). BDP showed good peak nature but the peak

Figure 4. Representative Pareto charts to show the influence of variables studied in the response of BDP and FFD using PB experimental design for HPTLC method.

Table IISystem Suitability Tests for HPLC (n ¼ 6)

Parameter BDP FFD

Retention time (min) 4.76 1.69Resolution 17.45Theoretical plates 7701 2342Tailing factor 1.22 1.65Injection repeatability (%RSD) 0.86 1.40

8 Parmar et al.

of FFD was not separated. Then acetonitrile was replaced by

methanol in the same ratio. Splitting was observed for both

peaks. Then acetonitrile, methanol and water were tried in dif-

ferent ratios but there were problem in peak shape of both drugs

and retention time of FFD was too long. Then the above mobile

phase in different ratios were tried along with change in pH

from 3.0 to 5.0 with the help of orthophosphoric acid. With 3.6

pH in 65:25:10 (v/v/v) ratio of methanol:acetonitrile:water, both

the drugs were separated within 5 min and have good peak

shapes. For stabilizing the pH, water was replaced by 25 mM

phosphate buffer (pH 3.6). Both the drugs showed typical peak

nature and peaks were symmetrical at 220 nm (Figure 3).

The proposed HPLC method utilizes an isocratic elution tech-

nique at room temperature with PDA detection for the simultan-

eous determination of both the drugs from rotacap formulation.

The mobile phase with pH 3.6 gives greater stability to the ana-

lytical column. The real advantage of the HPLC method is low re-

tention times: 1.69 and 4.78 min for FFD and BDP, respectively.

It reduces total run time, leads to low solvent consumption and

makes the method more economical.

Method validation

The goal of this study was to develop sensitive and robust chroma-

tographic methods for the simultaneous determination of BDP

and FFD from their combined marketed formulation. The pro-

posed methods were validated according to the ICH guidelines.

HPTLC method

The linearity of calibration graphs and adherence of the system

to Beer’s law was validated by high value of correlation coeffi-

cient and the SD for intercept value was ,2%. The repeatability

studies ensured precision of densitometric scanner and spotting

device. The % RSD for replicate sample solutions for intra- and

interday study are ,2.0% for FFD and BDP which met the ac-

ceptance criteria established for the RP-HPLC method. This con-

firms that the method is precise. The % RSD values show that the

proposed method provides acceptable intra- and interday vari-

ation of BDP and FFD. The signal-to-noise ratios of 3:1 and 10:1

were considered as LODs and LOQs, respectively.

In this paper, PB design has been chosen to test the robustness

of the HPTLC method. If the PB design is saturated, n trials enable

to determine n 2 1 factor effects. In the current study, seven

factors were tested with only eight experiments. The selection of

factors was based on observations during method development

and own experience. Each factor was studied at two levels. The

different levels for each factor were selected symmetrically

around the nominal value of the corresponding factor in the

Table IIIIntra- and Interday Precisions of BDP and FFD (n ¼ 3)

Drug HPTLC HPLC

Intraday precision Interday precision Intraday precision Interday precision

SD ofarea

%RSD

SD ofarea

%RSD

SD ofarea

%RSD

SD ofarea

%RSD

BDPa 64.40 0.51 109.36 0.87 6560.27 0.64 15083.29 1.17FFDb 7.60 0.34 19.59 1.00 666.88 0.47 1607.64 1.01

aAverage of three concentrations 120, 160, 200 ng/spot and 1.2, 2.4, 4.8 mg/mL for HPTLC and

HPLC, respectively.bAverage of three concentrations 3.6, 4.8, 6 mg/spot and 40, 80, 160 mg/mL for HPTLC and HPLC,

respectively.

Table IVRecovery Study of (a) HPTLC Method and (b) HPLC Method (n ¼ 3)

Excess drug added to the analyte (%) Theoretical content % Recovery % RSD

BDP(mg/spot)

FFD(ng/spot)

BDP FFD BDP FFD

(a) HPTLC method0 2.0 60 99.87 99.53 0.35 0.2460 3.2 96 99.74 99.81 0.24 0.40100 4.0 120 99.88 99.87 0.63 0.50140 4.8 144 99.91 99.82 0.61 0.36

(b) HPLC method0 40 1.2 99.98 99.78 0.27 0.3250 60 1.8 98.53 99.79 1.15 0.25100 80 2.4 99.99 99.66 0.28 0.47150 100 3.0 99.39 99.98 0.42 0.31

Table VEight Experiment PB Design to Examine the Seven Factors (A–G) Selected for Robustness Testing of HPTLC Method

Experiments Factors Responses

A B C D E F G % Recovery Rf values

BDP FFD BDP FFD

1 21 þ1 þ1 þ1 21 þ1 21 100.46 99.25 0.67 0.352 21 21 þ1 þ1 þ1 21 þ1 99.77 99.54 0.67 0.363 21 21 21 21 21 21 21 101.65 99.73 0.64 0.344 þ1 21 þ1 21 21 þ1 þ1 98.79 100.10 0.64 0.335 þ1 þ1 21 þ1 21 21 þ1 100.94 99.68 0.64 0.346 21 þ1 21 21 þ1 þ1 þ1 100.74 99.91 0.68 0.337 þ1 21 21 þ1 þ1 þ1 21 98.59 100.25 0.66 0.388 þ1 þ1 þ1 21 þ1 21 21 100.25 99.77 0.65 0.36

Responses Effects of factors Critical effect

A B C D E F G ME (a ¼ 0.05)

% Recovery (BDP) 21.01 0.90 20.66 20.42 20.62 21.01 20.18 1.729% Recovery (FFD) 0.34 20.25 20.22 20.20 0.18 0.20 0.06 0.525Rf values (BDP) 20.018 0.007 0.002 0.008 0.018 0.013 0.003 0.028Rf values (FFD) 0.0075 20.0075 0.0025 0.0175 0.0175 20.0025 20.0175 0.029

Sensitive and Robust Methods for BDP and FFD 9

original method. The limits of the factors studied were selected

according to error ranges which would be typically encountered

in an analytical laboratory. All absolute factor effects on the quan-

titative responses, i.e., percentage recoveries of BDP and FFD,

were found to be smaller than the corresponding critical effects,

i.e., ME (a ¼ 0.05) ¼ 1.729 for BDP and 0.525 for FFD. All absolute

effects on the response Rf values for both compounds were

smaller than their respective critical effects, i.e., ME (a ¼ 0.05)¼

0.028 for BDP and 0.029 for FFD. Concomitantly, graphical evalu-

ation employing Pareto charts was done. It may be more comfort-

able viewing the Pareto Chart that has the significant effects

selected. The Pareto graph (Figure 4) consists of bars with a

length proportional to the absolute value of the estimated effect,

divided by the pseudo standard error defined by Lenth (Lenth’s

Table VIEight Experiment 2421 Fractional Factorial Design to Examine the Four Factors (P–S) Selected for Robustness Testing of HPLC Method

Experiments Factors Responses Resolution

P Q R S % Recovery

BDP FFD

1 21 21 21 21 99.91 100.89 15.3782 þ1 21 21 þ1 99.84 99.59 15.3423 21 þ1 21 þ1 100.55 100.55 15.3844 þ1 þ1 21 21 99.70 99.38 15.2415 21 21 þ1 þ1 99.91 101.63 15.3156 þ1 21 þ1 21 99.92 101.60 15.1897 21 þ1 þ1 21 99.85 100.76 15.1498 þ1 þ1 þ1 þ1 99.91 99.20 15.343

Responses Effects of factors Critical effects

P Q R S PQ PR PS ME (a ¼ 0.05)

% Recovery of BDP 20.21 20.11 20.10 0.21 20.18 0.25 20.14 0.42% Recovery of FFD 21.02 20.95 0.70 20.41 20.35 0.22 20.68 1.66Resolution 20.03 20.03 20.09 0.11 0.05 0.06 0.02 0.15

Figure 5. Representative graphs to show the influence of variables studied in the response of BDP and FFD using 2421 experimental design for HPLC method.

10 Parmar et al.

PSE). All main effects and interaction terms are found to be statis-

tically insignificant as absolute values of main effects are below

the critical t-value (a ¼ 0.05). Thus, in all situations, the % recoveries

and Rf values for both the drugs are not significantly affected by

factor changes. The method was found to be robust.

HPLC method

The results of system suitability test (Table II) shows that the

optimized chromatographic conditions are adequate for simul-

taneous determination. The resolution of BDP and FFD peaks is

.1.5. Theoretical plates of BDP and FFD are .2,000, tailing

factor of BDP and FFD is ,2.0, % RSD of injection repeatability is

,2.0%. The recovery studies for BDP and FFD from pharmaceut-

ical formulation after spiking with additional standard drug

afforded recovery between 98 and 102%.

Robustness was assessed using experimental design approach.

The results of robustness trials were evaluated by half-normal

plots and statistical parameters. In half-normal plots, insignificant

effects that tend to have a normal distribution centered near

zero are normally distributed around the straight line, while sig-

nificant effects deviate from it. Ideally, when the selection of

statistically significant terms is complete, the Shapiro–Wilk

P-value must be .0.10, indicating that the remaining (unse-

lected) terms are normally distributed. In the current study,

Shapiro–Wilk P-value of all the response parameters are .0.10

indicates that all unselected terms are normally distributed on

straight line. It can be concluded from the half-normal plot that

no effects are statistically significant as they all are normally

distributed around the straight line. Although, we recommend

using the half-normal plot of effects to choose the statistically

significant effects, in some cases, a Pareto chart can also be very

valuable. The Pareto chart is a useful tool for showing others

the relative size of effects. There is standard t limits plotted on

the graph to identify significant effects. After the initial

selection of effects in half-normal plot, effects above the t-value

limit (t(a ¼ 0.05) ¼ 2.445) are possibly significant and should be

added if they are not already selected, effects below the t-value

limit are not likely to be significant. So, in present state, all main

effects and interaction terms are statistically in-significant. At the

studied ranges, the effects of the factors were not statistically sig-

nificant (a ¼ 0.05) for the response studied, recoveries of BDP

and FFD and resolution between BDP and FFD. All absolute

factor effects on the quantitative responses, i.e., percentage re-

coveries of BDP and FFD, and qualitative response, i.e., resolution

between BDP and FFD, were found to be smaller than the corre-

sponding critical effects. The % recoveries and resolution are

not significantly affected by factor changes.

Analysis of the marketed formulation

Both the drugs have good solubility in methanol; therefore,

methanol was selected for the extraction from rotacap formula-

tion. The formulation powder was sonicated with methanol for

25 min to ensure complete dissolution of both drugs. No inter-

fering peaks were found in the chromatogram, indicating that

formulation excipients did not interfere in the estimation of BDP

and FFD by the proposed chromatographic methods. The low %

RSD value indicated the suitability of this method for routine

analysis of FFD and BDP in pharmaceutical dosage form.

Content uniformity testing by HPLC

Due to the high precision of the proposed method and its ability

to rapidly estimate the concentration of the drugs in a single cap-

sules extract with sufficient accuracy, the method is ideally

suited for content uniformity testing which is a time-consuming

process when using conventional assay techniques. The steps of

the test were adopted according to the USP procedure (32). The

AV was calculated for each of the rotacaps and was found to be

smaller than the maximum LI.

Comparison between HPLC and HPTLC method

For four pairs of analysis, the Tcal was lower than the Ttab value

obtained from Student’s distribution table for a risk factor of 5%,

which showed that there was no statistically significant difference

between HPLC and HPTLC analytical methods. HPTLC has devel-

oped to an extent that separation and quantitation can provide

results that are comparable with another analytical method such as

HPLC. Today, HPTLC has firm place among various analytical techni-

ques as a reliable method for quantification at micro-, nano- and

even at picogram level, even when present in complex formulations.

Conclusion

The proposed HPTLC and HPLC methods provide precise, accur-

ate and reproducible quantitative analysis for the simultaneous

determination of BDP and FFD in rotacaps. Both the methods

were validated as per the ICH guidelines. The robustness of the

proposed methods was studied using DOEs and found to be

robust at deliberate changes made in experimental conditions.

Statistical tests indicate that the proposed HPTLC and HPLC

methods reduce the duration of analysis and appear to be

equally suitable for routine determination of BDP and FFD simul-

taneously in pharmaceutical formulation. The HPLC method is

also applicable for the content uniformity test of rotacaps. Both

the methods are suitable for routine analysis of BDP and FFD in

their commercial dosage forms. The methods are also suitable

and valid for application in quality control laboratories.

Supplementary material

Supplementary data are available at Journal of Chromatographic

Science online.

Table VIIStatistical Comparison of the Results Obtained by the Proposed HPLC and HPTLC Methods for the

Analysis of Formulation A and B

Parameters Formulation A Formulation B

BDP FFD BDP FFD

HPTLC HPLC HPTLC HPLC HPTLC HPLC HPTLC HPLC

Mean 100.07 99.97 99.54 99.96 99.85 100.06 99.68 100.01Variance 0.12 0.07 0.06 0.09 0.02 0.16 0.12 0.01d.f. 2 2 2 2t calculated 0.31 22.21 20.68 22.24t tabulated 2.92 2.92 2.92 2.92P-value* 0.39 0.08 0.28 0.08

*P-value ¼ 0.05.

Sensitive and Robust Methods for BDP and FFD 11

Acknowledgments

The authors thank Tripda Biotech Private Limited, Ahmedabad

for gift sample of beclomethasone dipropionate and Cadila

Healthcare Ltd., Ahmedabad for gift sample of formoterol fumar-

ate dihydrate. Authors also thank to Charotar University of

Science and Technology for providing the facilities for comple-

tion of the project.

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