Article

QbD-Based Development and Validation of a

Stability-Indicating HPLC Method for Estimating

Ketoprofen in Bulk Drug and Proniosomal

Vesicular System

Nand K. Yadav1, Ashish Raghuvanshi2, Gajanand Sharma3,4, Sarwar Beg3,

Om P. Katare3, and Sanju Nanda1,*

1Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India, 2Jubilant LifeSciences Limited, Noida, Uttar Pradesh 201301, India, 3Division of Pharmaceutics, University Institute of Pharmaceu-tical Sciences, UGC Centre of Advanced Studies, Panjab University, Chandigarh 160014, India, and 4FormulationResearch, Ipca Laboratories Limited, Kandivli Industrial Estate Ltd., Kandivli (W), Mumbai, Maharashtra 400067, India

*Author to whom correspondence should be addressed. Email: sn_mdu@rediffmail.com

Received 27 May 2015; Revised 8 August 2015

Abstract

The current studies entail systematic quality by design (QbD)-based development of simple, precise,

cost-effective and stability-indicating high-performance liquid chromatography method for estima-

tion of ketoprofen. Analytical target profile was defined and critical analytical attributes (CAAs) were

selected. Chromatographic separation was accomplished with an isocratic, reversed-phase chroma-

tography using C-18 column, pH 6.8, phosphate buffer–methanol (50 : 50v/v) as a mobile phase at a

flow rate of 1.0 mL/min and UV detection at 258 nm. Systematic optimization of chromatographic

method was performed using central composite design by evaluating theoretical plates and peak

tailing as the CAAs. The method was validated as per International Conference on Harmonization

guidelines with parameters such as high sensitivity, specificity of the method with linearity ranging

between 0.05 and 250 µg/mL, detection limit of 0.025 µg/mL and quantification limit of 0.05 µg/mL.

Precision was demonstrated using relative standard deviation of 1.21%. Stress degradation studies

performed using acid, base, peroxide, thermal and photolytic methods helped in identifying the

degradation products in the proniosome delivery systems. The results successfully demonstrated

the utility of QbD for optimizing the chromatographic conditions for developing highly sensitive

liquid chromatographic method for ketoprofen.

Introduction

Ketoprofen (KT), a propionic acid derivative (Figure 1), belongs to thefamily of nonsteroidal anti-inflammatory drugs, with a pKa of 4.45.KT is clinically effective in dysmenorrhea, rheumatic and traumaticpain, postoperative pains of orthopedic origin and inhibits inflamma-tory mediators in human dental pulp cells (1, 2). Pharmacological ac-tion of KT is associated with the inhibition of prostaglandin synthesisand its anti-inflammatory effects are probably due to the inhibition ofcyclooxygenase-1 and cyclooxygenase-2 (3, 4).

Owing to the associated side-effects, oral dosage form of KT is los-ing interest among the health practitioners. Novel dosage forms likevesicular delivery systems for topical treatment are gaining interestnowadays, which do not cause oral side-effects and ensure better avail-ability of KT at specific site for longer time with controlled effect. Theliterature revealed the development of many novel carriers like vesicu-lar delivery systems viz. liposomes, elastic liposomes and proliposomes(5–7). Our research group has developed KT proniosome vesiculardelivery system for periodontitis treatment. Proniosomes are semisolid

Journal of Chromatographic Science, 2016, Vol. 54, No. 3, 377–389doi: 10.1093/chromsci/bmv151

Advance Access Publication Date: 29 October 2015Article

© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com 377

liquid crystal/powder products which upon hydration are easilyconverted into niosomes, hence serve as a ready-to-form niosomeformulation, which overcomes the drawbacks associated with nio-some formulations. Owing to their small size and bilayer structure,proniosomes enhance absorption of drugs along with prolonged dura-tion of action (8).

Several methods have been reported for the determination of KT invarious formulations such as tablets, capsules, creams, topical gel andbiological samples, such as blood, plasma and urine. The methods re-ported in the literature include spectrophotometric (9, 10) and liquidchromatographic methods (11–13). The United States Pharmacopoeiaalso reports high-performance liquid chromatography (HPLC) meth-od for the assay of KT in capsule formulation (14). The optimizationof reported methods was not discussed in detail and thus there is still ascope of improvement in method optimization part. None of the re-ported methods employed analytical quality by design (AQbD) toolfor systematic development, thus lacks in comprehensive understand-ing of the effect of critical chromatographic parameters on methodperformance. Moreover, validated HPLC method for the estimationof KT in proniosome vesicular delivery system has not been reportedso far.

Traditional approach of method development comprisestrial-and-error approach and by varying one-factor-at-a-time. Thisapproach results in a large number of experiments and lacks inunderstanding of critical parameters. Recent use of AQbD in ana-lytical method is gaining interest nowadays, which emphasizes onscience- and risk-based understanding of critical parameters affect-ing method performance and results in a robust method (15–17).AQbD, a systematic approach in method developments utilizes de-sign of experiments, which provide assurance of quality and helps inthorough understanding of possible interactions among the methodvariables (18, 19). This systematic approach involves a series ofsteps starting from defining analytical target profile (ATP) andcritical analytical attributes (CAAs), screening of critical methodparameters (CMPs) and optimization using experimental designs.The optimization process results in analytical design space alsoknown as analytical method working space (16). AQbD in analyt-ical method development has been effectively employed in recentyears and results in robust analytical methods for estimationof active in bulk drugs, pharmaceutical dosage forms and in biolog-ical fluids (20–23). These reported methods cannot be extendedto analyze KT in the present proniosome vesicle formulation,due to the difference in formulation components and thus produceinterference.

The objective of the present study was to develop a precise, robustand stability-indicating HPLC method for the determination of KT inproniosome vesicle formulation employing AQbD approach. More-over, the proposed method has distinct advantages over previously re-ported methods with its cost effectiveness by using methanol andphosphate buffer, wide linearity range, short run time and is devoidof any interference of formulation excipients.

Methods

Chemicals and reagents

KT standard (99.3% purity) and API were kindly supplied by BECChemicals, Mumbai (India), and were used as such without furthertreatment. Methanol (HPLC grade) was purchased from SD FineChemicals, India. Sodium hydroxide, hydrochloric acid and hydrogenperoxidewere obtained from Fischer Scientific, India. Ortho phospho-ric acid 88% was purchased from Merck, India. HPLC-grade waterwas obtained from Milli-Q system (Millipore, Milford, MA, USA)and was used to prepare all solutions. All other chemicals and solventsused were of analytical grade.

Preparation of KT proniosomes

KT proniosome formulation was prepared by slightly modifying themethod reported in the literature (24). Precisely, the drug, surfactant(span 80, cholesterol and soya lecithin) and oleic acid were taken in aclean and dry wide mouth small glass tube and small amount of buta-nol was added to it. Widemouth glass tubewas heated on awater bathmaintained at 60–70°C until the entire solid portion dissolved. Aque-ous phase (pH 6.8 phosphate buffer) was added to it and the mixturewas heated again on a water bath till clear solution was obtained. Theobtained solution was cooled and overnight storage of the solution ledto the formation of proniosome gel. The obtained gel was preserved inclosed air tight container for further characterization.

Instrumentation

Two different brands of HPLC systems were used, aWaters HPLC sys-tem (Waters 2695 separation module), equipped with Waters 2996photodiode array (PDA) detector, a column oven and a quaternarypump system, the data were acquired using the Empower software.The second system used was an Agilent 1100 system equipped witha degasser (model G1379A), quaternary pump (model G1311A), anautosampler (model G1329A), a thermostat heater (model G13308),a variablewavelength detector (model G1314A) and data analysis wasperformed using Agilent Chemstation for LC systems software Ver-sion B.04.02 (Agilent Technologies, Santa Clara, CA, USA).

Chromatographic conditions

A reversed-phase HPLC separation was carried out using KromasilC-18 column (150 mm × 4.6 mm, 5 µm packing) and an isocratic elu-tion with mobile phase consisted of a mixture of methanol and phos-phate buffer (13 mM KH2PO4 adjusted to pH 6.5 with H3PO4) in50 : 50 (v/v) ratio at a flow rate of 1.0 mL/min. Sample detectionwas monitored at a wavelength of 258 nm with an injection volumeof 10 µL. The sample and column temperature were maintained at25 and 30°C, respectively. The mobile phase was filtered with0.45-µm nylon filter and degassed in ultrasonic bath prior to use.Peak purity values were obtained directly from spectral analysis reportobtained using the instrument software (Waters HPLC system usingEmpower software). The forced degradation samples were analyzedusing a PDA detector in scan mode covering the wavelength rangeof 200–400 nm.

Defining ATP and CAAs

The systematic development of analytical method starts by definingATP, which is the essential element of AQbD approach and formsthe basis for design of an analytical method. The ATP includes all at-tributes that are needed to ensure the quality characteristic and pur-pose of analytical method. Table I describes various elements ofATP, defined to obtain an efficient chromatographic method for KTestimation. Identification of CAAs from ATP is based on the critical

Figure 1. Chemical structure of KT, 2-(3-benzoylphenyl) propionic acid.

378 Yadav et al.

contribution of that particular attribute on the performance and qual-ity of analytical method. Out of various attributes, only two are con-sidered as CAAs, i.e., number of theoretical plates (indicator ofmethod performance and suitability) and peak tailing, which is con-sidered as the indicator of method efficiency.

Preparation of stock and standard solutions

A standard stock solution of KT (250 µg/mL) was prepared by accu-rately weighed amount (∼50 mg) and diluted using mobile phase intoa 200-mL volumetric flask. The standard solution of KTwas preparedusing 5 mL of the above prepared standard stock solution and dilutedup to 50 mL in a volumetric flask using mobile phase, which gave afinal standard solution concentration equal to 25 µg/mL.

Preparation of KT proniosome samples

About 2 g of the KT proniosome gel was accurately weighed andtransferred to 200-mL volumetric flask, using mobile phase and son-icated for 15 min to enable complete extraction of KT. The solutionwas filtered, 5 mL of filtrate was transferred to 50 mL volumetricflasks and diluted using mobile phase to yield a concentration equalto 25 µg/mL. These solutions were filtered through a 0.45-µm nylonmembrane filter before injections.

Taguchi design for factor screening

Initially, as a part of the QbD-based method development exercise, theIshikawa fish-bone diagram was drawn employing the Minitab 17software (M/s Minitab Inc., Philadelphia, USA). A Taguchi experi-mental design was employed to evaluate the effects of seven indepen-dent chromatographic parameters on the two defined CAAs,theoretical plates and peak tailing. The design comprises of eight ex-perimental runs and helps in screening of factors by evaluating theirmain effect to get CMPs. Table II comprises details of independent pa-rameters and their studied ranges and studied responses (CAAs).

Method development using response surface

methodology

Two method parameters were selected based on their significant effecton selected CAAs and considered as CMPs, as an outcome of screen-ing study. Mobile phase ratio (X1) and pH of mobile phase buffer (X2)were selected for further optimization. A central composite design(CCD) with α = 1 was applied to evaluate the main and interaction ef-fect of the selected two factors. A total of 13 experiments were con-ducted as per the design matrix summarized in Table III. All otherparameters were kept fixed at their optimum levels during the experi-mentation and a standard concentration of 25 µg/mL was maintained.Analysis of the experimental data was performed against the definedCAAs (theoretical plates and peak tailing). Statistical analysis of thedata obtained from factor screening and method development optimi-zation were performed using the Design-Expert® software version9.0.4 (Stat-Ease Inc., Minneapolis, USA). Numerical and graphicaloptimization was carried out to get an optimal “analytical designspace” region.

Method validation

The optimized chromatographic method was validated for linearityrange, accuracy, precision, limit of detection (LOD), limit of quantita-tion (LOQ) and robustness according to the International Conferenceon Harmonization (ICH) Q2(R1) guidelines (25).

System suitabilitySystem suitability test of the chromatography system was performedby injecting six replicate injections of standard solution 25 µg/mL.Prior to sample analysis, percent relative standard deviation (%RSD) of standard area and retention time for six suitability injectionswere determined and accepted below 2.0%. The acceptancecriteria for tailing factor of <2.0 and theoretical plates >2,000 weremaintained.

Table I. ATP of Liquid Chromatographic Method for KT

ATP elements Target Justification

Target analyte KT Analytical method development for the detection of KT, activeanalyte in samples

Target sample API/proniosome vesicles Development of an analytical method for estimation of KT in APIand proniosome vesicle formulation for routine and stabilityanalysis

Sample form API is in solid formProniosome vesicles in gel form

KT exists in solid form; however, KT is entrapped in proniosomevesicle gel to give benefit to the patients

Analytical technique Reverse-phase liquid chromatography Reverse-phase liquid chromatography is a more reliable and widelyused technique, where hydrophobic stationary phase andhydrophilic mobile phase result in better retention for the majorityof the lipophilic drugs like KT (log P 3.3)

Instrument usage HPLC equipped with quaternary pumpsystem and PDA detector

Quaternary pump results in precise mixing of mobile phase solvents,whereas PDA results in detection of probable degradants atvariable wavelength

Sample preparation API/proniosome vesicle to be converted toliquid state

To detect and quantify analyte, samples must be converted to liquidform for its miscibility with mobile phase

Target method Assay The developed method should be capable of assaying KT in bulkform and pharmaceutical dosage form (proniosome vesicles) forroutine and stability/stress samples

Analytical method qualityattributes

Theoretical plate count To develop a quality method, these attributes should meet thecompendial or other applicable quality standardsRetention time

Peak tailingResolution

Estimating Ketoprofen in Bulk Drug and Proniosomal Vesicular System 379

Linearity and rangeCalibration curve of the developed method was prepared by serialdilution of stock solutions in concentrations ranging between 0.05and 250 µg/mL, which corresponds to 0.2–1,000% of the standardpreparation concentration. The calibration curve was constructedby plotting the peak area against drug concentration using linearregression analysis.

AccuracyAccuracy of analytical method was demonstrated by using knownquantities of KT spiked at three different levels (12.5, 25 and37.5 µg/mL). Stock solution of KT (250 µg/mL) was prepared by

accurately weighed amount (∼50 mg) and further spiked at three lev-els. Accuracy was performed at ∼50, 100 and 150% of the theoreticalKT concentration level in sample. The percentage recovery of theadded drug was calculated by comparing % RSD of the peak areaof test sample with that of the standard solution.

PrecisionThe precision of an analytical method depicts the closeness of agree-ment (degree of scatter) between a series of measurements using theprescribed conditions. Precision was expressed in terms of % RSDfor peak area or corresponding assay value.

System precisionSix replicate injections of standard solution were prepared, injected inthe HPLC system and analyzed by proposed method to assess thesystem precision.

Method precision. Six samples of a single batch of KT proniosomeformulation were prepared and analyzed by proposed method toassess the method precision.

Intermediate precision. Intermediate precision was performed byinjecting six preparations in duplicate. KT proniosome formulationpreparations were analyzed using different HPLC system, differentanalyst and different brand of C-18 column on a different day.

LOD and LOQLOD and LOQwere determined based on signal-to-noise ratios of an-alytical responses of 3 : 1 and 10 : 1, respectively.

RobustnessThe robustness of an analytical procedure refers to its ability to remainunaffected by small and deliberate variations in method parametersand provides an indication of its reliability for routine analysis. Ro-bustness of the method was studied by evaluating system suitabilityparameters after modifications in system operating parameters such

Table II. Matrix of Taguchi Design for Screening of Method Parameter Along With the Studied Levels

Run Mobile phase ratio pH of mobile phase buffer Flow rate Injection volume Column oven temperature Column dimension Wavelength

1 Low High High Low Low High High2 Low High High High High Low Low3 Low Low Low Low Low Low Low4 High High Low Low High High Low5 Low Low Low High High High High6 High Low High Low High Low High7 High High Low High Low Low High8 High Low High High Low High Low

Chromatographic method variables Levels

Low High

Mobile phase ratio (methanol : buffer) 40 : 60 60 : 40pH of mobile phase buffer 6.3 6.7Flow rate (mL/min) 0.8 1.2Injection volume (µL) 5 15Column oven temperature (°C) 25 35Column dimension (mm) 150 250Wavelength (λmax) 253 263

Response Target

Theoretical plates Maximum (>2,000)Peak tailing Minimum (≤2)

Table III. Two-Factor CCD Matrix Along with Studied Levels

Run Factor code

X1 X2

1 0 02 0 03 1 14 0 05 1 −16 −1 −17 −1 18 0 09 0 010 1 011 0 112 0 −113 −1 0

Codes translation in actual values

Code levels −1 0 1

X1: mobile phase ratio 40 : 60 50 : 50 60 : 40X2: pH of mobile phase buffer 6.3 6.5 6.7

380 Yadav et al.

as injection volume (±50%), flow rate (±10%) and column tempera-ture (±2°C).

Solution stabilityStability of KT in standard and proniosome sample solution was eval-uated by injecting the samples at a time interval of 0, 6, 24, 48 and72 h. The results were compared in terms of % change in assayvalue for all studied standard and sample preparation with that offresh preparations.

Forced degradation studiesForced degradation studies were performed to evaluate the stability in-dicating and specificity property of the developed assay method andthese studies were conducted as per ICH and other recommended con-ditions (26, 27). Standard/sample preparations for stress studies wereprepared at a concentration of 0.2 mg/mL and subjected to variousstress conditions. All samples were then diluted accordingly to give afinal concentration of 25 µg/mL neutralized wherever applicable andfiltered before injection. Control samples were also prepared and usedas a control during analysis.

Oxidation studies. Solutions for oxidation studies were prepared using3% H2O2, protected from light, stored at room temperature for 12 hand heated at 80°C for 5 h. Samples were withdrawn at specified timeintervals and diluted as previously described.

Acid degradation studies. Solutions for acid degradation studies wereprepared using 1 M hydrochloric acid, protected from light, stored atroom temperature for 12 h and heated at 80°C for 5 h. Samples wereneutralized and diluted as previously described.

Alkali degradation studies. Solutions for base degradation studieswere prepared using 1 N sodium hydroxide, protected from light,stored at room temperature for 12 h and heated at 80°C for 5 h. Sam-ples were neutralized and diluted as previously described.

Thermal degradation. Samples were exposed to dry heat at 80°C in anoven for 12 h. Samples were withdrawn, cooled and diluted as previ-ously described.

Photodegradation. Samples solutions were exposed to UV light for12 h with illumination of 7,500 Lux meter with UV radiation at320–400 nm in UV light chamber. Samples werewithdrawn and dilut-ed as previously described.

Peak purityPeak purity analysis is designed to detect the presence of an impuritythat is coeluting with the analyte peak. Peak purity of KT and its deg-radation impurities were determined using Waters HPLC system (Em-power software) and PDA detector. Peak purity was calculated interms of purity ratio, which is calculated using the purity angle andpurity threshold. Ratio of purity angle and purity threshold shouldbe <1, only then the peak is considered to be spectrally pure.

Results

Preliminary studies and factor selection

In search of a simple, stability-indicating and cost-effective LCmethodfor estimation of KT in standard and proniosome vesicular formula-tion, a preliminary study was carried out. Mobile phase composed

of organic solvent (methanol and acetonitrile) and a variety of aque-ous solutions of inorganic salt having different pH (acetate buffer andphosphate buffer), at variable flow rates (0.8 to 1.2 mL/min) were in-vestigated during the preliminary studies of the analytical method de-velopments and system suitability parameters were evaluated for eachrun. KT standard, KT proniosome and stressed samples with KromasilC-18 column and PDA detector were used during initial investigationtrials. The preliminary studies suggested the selection of methanol andpH 6.5 phosphate buffer as a mobile phase, as it showed better chro-matographic separation.

Taguchi design for factor screening

The probable factors contributing to the chromatographic methodperformance are summarized in Ishikawa fish-bone diagram (Fig-ure 2). Taguchi experimental design was used for screening of factorsidentified using risk assessment studies. Out of several factors, theemployed design studied the main effects of seven factors affectingtheoretical plates and peak tailing of KT peak identified as CAAs.The main purpose of this screening study was to identify the signifi-cant main effect of studied factor with minimum possible experimen-tation. Model analysis was performed using first-degree polynomialequation. The influence of studied factors on method attributes wasstudied using pareto charts presented in Figure 3, where the paretoranking of most influential factors was given. Two factors namely,mobile phase ratio and pH of mobile phase buffer showed significanteffect (P < 0.05) on CAAs (theoretical plates and peak tailing). Mo-bile phase ratio and pH of mobile phase buffer showed a positive ef-fect on theoretical plates. As ratio and pH of mobile phase increase,theoretical plate count also increases. However, inverse relationshipwas observed for both the parameters on peak tailing. On the basis ofthe screening study, other parameters were kept constant for furtheroptimization of two selected CMPs. Flow rate and injection volumewas kept fixed at 1.0 mL/min and 10 µL, respectively. Furthercolumn oven temperature, column dimension and wavelength werefixed at 30°C, 150 mm and 258 nm, respectively. However, mobilephase ratio and pH of mobile phase buffer were further optimizedto determine any main and interaction effect on theoretical platesand peak tailing.

Method development using response surface

methodology

CCD design was employed in the present analytical method optimiza-tion study. Data analysis was carried out using second-degree polyno-mial mathematical model and evaluated for main and interaction

Figure 2. Ishikawa fish-bone diagram showing probable causes affecting the

efficiency of target analytical method of KT. This figure is available in black

and white in print and in color at JCS online.

Estimating Ketoprofen in Bulk Drug and Proniosomal Vesicular System 381

effect of factors. The quadratic Equation (1) generated using statisticalanalysis of software established the relation between CMPs andCAAs.

Y ¼ b0 þ b1X1 þ b2X2 þ b3X1X2 þ b4X21 þ b5X2

2 ð1Þ

where b0 is the intercept, b1–b5 represents the regression coefficients,values of these coefficients were calculated based on the interaction be-tween response and factors. X1 and X2 represent the coded CMPs, Yrepresents CAA. Table IV portrays coefficient value of polynomialequation and model P-value for method CAAs.

Theoretical platesTheoretical plate count is an important parameter considered as CAA,as this indicates method performance and suitability. Theoretical platecount should be higher (>2,000) for better method performance. Forthis method, attribute quadratic model was found significant(<0.0001), whereas lack of fit was insignificant (P = 0.1298). X1, X2,X2

1 and X22 are the significant model terms, whereas X1X2 terms is in-

significant. Model diagnosis plots are presented in Figure 4, includesnormal and residual versus run plot indicates suitability of the model.Contour plots were also studied to visualize the effects of the factorsand their interactions on the response. Figure 5 portrays 3D and 2Dcounter plots showing effect of mobile phase ratio and mobile phasebuffer pH on theoretical plates. Curvatures in contour plot showednonlinear or interaction effect of factors on theoretical plates.A curvilinear increasing trend was observed for mobile phase ratio,which showed higher theoretical plate count at higher levels. However,a curvature effect was observed for mobile phase pH at higher and lowlevels. Therefore, higher level of mobile phase ratio and optimum levelof pH were recommended to achieve higher theoretical plates.

Peak tailingDesired peak tailing of the compound peak results in increasedmethod efficiency. It was also considered an important parameterand selected as CAA of the method. Quadratic model was found sig-nificant (P < 0.0001) for peak tailing, whereas lack of fit was insignif-icant (P = 0.7079). X1, X2 and X2

2 are the significant model terms,whereas X1X2 and X2

1 terms are insignificant. Suitability of modelwas also proved by model diagnosis plots presented in Figure 6, in-cludes normal and residual versus run plots. Study of 3D and 2Dcounter plots presented in Figure 7 showed curved effects of mobilephase ratio and mobile phase buffer pH on peak tailing. A decreasingcurvature trend was observed for both mobile phase ratio and mobilephase pH, which showed higher peak tailing at lower levels. The de-sired peak tailing of nearly 1 was achieved at higher levels of bothCAAs.

Optimized method conditionsTo obtain optimummethod conditions, numerical optimization meth-ods were applied based on the specified goals and boundaries for eachresponse. The desired goals include maximum theoretical plate countand minimized peak tailing close to 1. The optimized method condi-tions for KT estimation were obtained, which include mobile phasecomposition containing a mixture of methanol and phosphate buffer(13 mMKH2PO4) in 50 : 50 (v/v) ratio and mobile phase buffer pH of6.5 with desirability of 1. Figure 8 shows overlay plot with optimumregion as a design space and selected method conditions were repre-sented using flag.

Method validation

Linearity and rangeLinearity of the developed method was confirmed by plotting linearitycurve for concentrations ranging 0.05–250 µg/mL, which correspondsto 0.2–1,000% of the standard preparation concentration. The cali-bration curve (Figure 9) was constructed by plotting the peak areaagainst the concentration using linear regression analysis. The correla-tion coefficient (R2 = 0.999) obtained above for the linear regressionline demonstrates the excellent relationship between peak area andthe concentration.

AccuracyAccuracy of analytical method performed at 50, 100 and 150% of thestandard KT concentration showed recovery between 98.8 and101.2%. Data shown in Table V indicate that the developed methodhas high level of accuracy with % RSD ranging from 0.31 to 0.41%.

Table IV.Regression Coefficients of Polynomial Equation Alongwith

P-value

Factors Theoretical plates Peak tailing

Coefficient P-value Coefficient P-value

Intercept 5512.72 1.10X1 98.83 <0.0001 −0.045 <0.0001X2 18.00 0.0225 −0.068 <0.0001X1X2 12.75 0.1355 −0.015 0.0353X2

1 28.47 0.0166 0.013 0.0971X2

2 −47.03 0.013 0.023 0.0121

Figure 3. Pareto chart showing effect of factors on theoretical plates (A) and peak tailing (B). This figure is available in black and white in print and in color at JCSonline.

382 Yadav et al.

Precision.System precision. Analytical data from six replicate injections of stan-dard solution showed%RSD value of 0.31%.Data shown in Table VIindicate an acceptable level of precision for the analytical system as theRSD observed was 0.31%.

Method precision.Analytical data from six samples of a single KT pro-niosome formulation batch showed recovery of 98.1% with a % RSDvalue of 0.96%. Table VI represents method precision data. % RSDbelow 1% depicts higher degree of method precision.

Intermediate precision. Intermediate precision data representedin Table VI showed overall % RSD value of 1.21% between thetwo sets of data. Further, the percent recovery of KT rangingbetween 96.4 and 98.1% confirmed higher degree of intermediateprecision.

LOD and LOQThemethod observed LOD and LOQ values of 0.025 and 0.05 µg/mL,respectively, indicates higher sensitivity of developed method for KTquantification.

Figure 4. Model diagnosis graphs of the experimental data for theoretical plates. Normal plot of residuals: The normal probability plot indicates whether the

residuals follow a normal distribution, in case the points follow a straight line with some moderate scatter. If the scatter pattern shows a “S-shaped” curve, it

indicates that a transformation of the response may provide a better analysis. Residuals versus Run: Plot of the residuals versus the experimental run order

allows checking for lurking variables that may have influenced the response during the experiment and should show a random scatter. Trends indicate a

time-related variable lurking in the background. Blocking and randomization provide insurance against trends ruining the analysis. This figure is available in

black and white in print and in color at JCS online.

Figure 5. 3D (A) and 2D (B) contour plots showing effect of mobile phase ratio (X1) and mobile phase buffer pH (X2) on theoretical plates. This figure is available in

black and white in print and in color at JCS online.

Estimating Ketoprofen in Bulk Drug and Proniosomal Vesicular System 383

RobustnessThe influence of each variable, such as injection volume, flow rate andcolumn temperature caused insignificant changes in the theoreticalplates, peak tailing, retention time and peak area RSD. The insensitiv-ity toward deliberate minor changes in the method parameters demon-strated robustness of the systematically developed analytical method.

Solution stabilityThe corresponding chromatograms of KT in standard and pronio-somes sample showed no peak of degradation products and also

there was no significant change in the peak area. The assay resultsof the sample and standard were found within ±2.0% comparedwith fresh solution.

Forced degradation studiesThe degradation of KT under various stress conditions was evaluated,typical chromatograms obtained from stressed studies are presented inFigure 10. Details of degradation products, % degradation and peakpurity are illustrated in Table VII. Mainly two degradation productswere seen from most of the conditions and reported at ∼14 and

Figure 6. Model diagnosis graphs of the experimental data for peak tailing. Normal plot of residuals: The normal probability plot indicates whether the residuals

follow a normal distribution, in case the points follow a straight line with somemoderate scatter. If the scatter pattern shows a “S-shaped” curve, it indicates that a

transformation of the response may provide a better analysis. Residuals versus Run: Plot of the residuals versus the experimental run order allows checking for

lurking variables that may have influenced the response during the experiment and should show a random scatter. Trends indicate a time-related variable lurking in

the background. Blocking and randomization provide insurance against trends ruining the analysis. This figure is available in black and white in print and in color at

JCS online.

Figure 7. 3D (A) and 2D (B) contour plots showing the effect ofmobile phase ratio (X1) andmobile phase buffer pH (X2) on peak tailing. This figure is available in black

and white in print and in color at JCS online.

384 Yadav et al.

16 min, whereas the KT peak was observed at ∼5.9 min. KT is moresusceptible to oxidative degradation, as the maximum degradationwas observed with 3% H2O2 heated at 80°C for 12 h, i.e., 5.2%.

Acid/base and thermal degradation. Acidic treatment in 1 M HCl for12 h resulted in no degradation, whereas treatment with 1 M HClheated at 80°C for 5 h resulted in 0.4%degradation and the degradantpeak was observed at 16.46 min. Treatment with 1 N NaOH for 12 hproduced no degradation; however, 1 NNaOHheated at 80°C for 5 hresulted in two degradation products at 14.02 and 16.19 min and thetotal degradation was 1.0%. There was no noteworthy degradation ofKT exposed to dry heat at 80°C for 12 h and the KT was found stableat this condition.

Photolytic and oxidative degradation.Degradation of KT (2.0%) wasobserved on exposure to UV light for 12 h and the single degradationpeak appeared at 16.24 min. In oxidation studies, mild degradation, i.e., 0.4% was observed when treated with 3% H2O2 for 12 h and asingle degradation peak was observed at 16.26 min. Introduction ofheat in the peroxide treatment (3% H2O2 heated at 80°C for 12 h)sped up the degradation and a total 5.2% degradation was reportedand the degradation was contributed by two degradation products re-ported at 14.35 and 16.59 min.

Figure 8.Overlay plot showing optimal analytical design space along with values of method parameters and CAAs of selected point as a flag. This figure is available

in black and white in print and in color at JCS online.

Figure 9. Linear calibration curve of KT. This figure is available in black and

white in print and in color at JCS online.

Table V. Accuracy Data

Level (%) Parameter KT (% recovery)

50 Mean 101.2% RSD 0.36

100 Mean 99.2% RSD 0.41

150 Mean 98.8% RSD 0.31

Table VI. Precision Studies of the Developed Method

Precision type Parameter Studied response

System precision Mean 948,853 (peak area)% RSD 0.31

Method precision Mean 98.1 (% assay)% RSD 0.96

Intermediate precision Mean 97.3 (% assay)% RSD 1.21

Estimating Ketoprofen in Bulk Drug and Proniosomal Vesicular System 385

Selectivity/specificity. The peak purity of KT peak was calculatedusing purity angle and purity threshold. Purity angle is defined as ameasure of the spectral heterogeneity of a peak based on the compar-ison of spectra over the entire peak, using the spectral contrast angle,

whereas purity threshold is the maximum permissible angle of peakbelow which the peak is considered to be pure. Ratio of purity angleand purity threshold below 1 confirmed the purity of KT peak. Thedegradation products were well separated/resolved from KT peak

Figure 10. Typical HPLC chromatograms of untreated formulation (A); HCl treated at 80°C for 5 h (B); NaOH treated at 80°C for 5 h (C); H2O2 treated for 12 h (D); H2O2

treated at 80°C for 5 h (E); UV exposure for 12 h (F).

386 Yadav et al.

and no placebo interference was observed; moreover, the peak purityvalue established that KT peak is homogenous and there are no coe-luting peaks, established the stability-indicating power of the method.The selectivity/specificity of the newly developed method was assessedusing the stress studies, and the results of the stress studies indicatehigh degree of selectivity of the method. The degradation patternwas found to be similar for KT and KT proniosome formulation.

Analysis of in-house-developed novel proniosome

formulation

Systematically developed HPLC method was used for the quantitativedetermination of KT in bulk drug and in-house-developed proniosomeformulation. The analytical method was validated for different param-eters, thus applied for the estimation of the drug in pharmaceutical dos-age forms. The percent recovery was 99.7%, and CAAs, like theoretical

Figure 10. Continued

Estimating Ketoprofen in Bulk Drug and Proniosomal Vesicular System 387

plates and peak tailing, were found to bewell within the acceptable lim-its. The absence of any unwanted peaks in the chromatograms suggest-ed noninterference of formulation excipients with the main analytepeak. Moreover, forced degradation studies established the peak purityof KT peak in the presence of degradants. This suggests that no otherpeak is eluting with main peak and showed lack of interference. The re-sults indicated the method usefulness for routine and stability sampleanalysis of KT in proniosome formulation.

Discussion

In a nutshell, current studies utilized the systematic approach of AQbDprinciples for successful establishment of a liquid chromatographicmethod for KT estimation in bulk drug and proniosome vesicle formu-lation. ATPs of the chromatographic method were defined at initialstage; this results in selection of theoretical plate count and peak tail-ing as potential CAAs. However, usage of fish-bone diagram is usefulin identifying probable chromatographic parameters for screeningstudies. Seven chromatographic parameters were employed for furtherscreening of their main effects on the identified CAAs using Taguchidesign. Two factors that showed significant impact of CAAs were fur-ther optimized using CCD of response surface methodology by study-ing their main and interaction effects. Optimized chromatographicconditions were selected by numerical optimization using desirabilityfunction. Systematically developed method was successfully validatedfor linearity, accuracy, precision, robustness and specificity parame-ters. The developed method is cost-effective in terms of usage of low-cost organic solvents (methanol) in mobile phase, whereas most of thereported methods used acetonitrile as an organic solvent.

Conclusion

A new stability-indicating simple, cost-effective, accurate, robust andspecific HPLCmethod was developed. The method is suitable for rou-tine analysis of bulk drug and proniosome formulation and can be uti-lized for the analysis of other vesicular delivery systems also. Simplicityof the method can be helpful in saving money and time for small lab-oratories with wide linear range and accuracy. Forced degradationstudies in various stress conditions resulted in a selective/specific andstability-indicating method. The proposed method has no interferenceof degradation products and has the ability to separate the degrada-tion products from the KT peak. The developed method can also beused for the analysis of stability samples to predict the shelf life ofthe pharmaceutical product. The developed method successfully

demonstrates the applicability of AQbD methodology in the develop-ment of a liquid chromatographic method for the estimation of KT.

Authors’ contributions

N.K.Y. and A.R. have performed the experimental work and prepareddraft of the manuscript. S.N. and O.P prepared the guidelines and su-pervised this work. K.G.S. and S.B. have read, checked and modifiedthe manuscript. All authors read and approved the final manuscript.

Acknowledgments

We gratefully thank BEC Chemicals, Mumbai (India), for providing the giftsample of KT and Jubilant Life Sciences Ltd., Noida, India, for providing thelaboratory facility for the research work.

Conflict of interest statement. The authors declare that they have no competinginterests.

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Control sample No treatment – – 101.4 0.151 0.349Acid degradation 1 M HCl, 12 h – – 98.0 0.169 0.355

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