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Development and Validationof RP-HPLC Method forSimultaneous Estimationof Three-ComponentTablet FormulationContaining Acetaminophen,Chlorzoxazone, andAceclofenacR. Joshi a & R. Sharma aa School of Pharmacy, Devi Ahilya Vishwavidyalaya,Takshshila Campus, Indore, IndiaPublished online: 04 Dec 2008.

To cite this article: R. Joshi & R. Sharma (2008) Development and Validation of RP-HPLC Method for Simultaneous Estimation of Three-Component Tablet FormulationContaining Acetaminophen, Chlorzoxazone, and Aceclofenac, Analytical Letters,41:18, 3297-3308, DOI: 10.1080/00032710802515086

To link to this article: http://dx.doi.org/10.1080/00032710802515086

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PHARMACEUTICAL ANALYSIS

Development and Validation of RP-HPLCMethod for Simultaneous Estimation of Three-

Component Tablet Formulation ContainingAcetaminophen, Chlorzoxazone, and Aceclofenac

R. Joshi and R. SharmaSchool of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshshila Campus,

Indore, India

Abstract: The present work describes a simple reversed-phase high-performanceliquid chromatographic method that has been developed and validated for simul-taneous estimation of acetaminophen, chlorzoxazone, and aceclofenac in tabletdosage form. The estimation was carried out on an Luna C18 (5 mm� 25 cm�4.6 mm i.d.) column using a mixture of buffer, methanol, and acetonitrile in theratio 215:130:155 with final pH of 6.5 as a mobile phase, at a flow rate of1.5 ml=min. Ultraviolet (UV) detection was performed at 275 nm. Total run timewas 10 min; these three drugs (acetaminophen, chlorzoxazone, and aceclofenac)were eluted at the retention times of 2.055, 5.096, and 7.605 min respectively.The method was validated for accuracy, precision, linearity, specificity, and sen-sitivity as per ICH norms.. From the validation study it was found that themethod is specific, rapid, accurate, precise, and reproducible. Calibration curves

Received 9 June 2008; accepted 10 September 2008.The authors are thankful to Zest Pharma, 275 Sector-F, Sanwer Road, Indore,

India, for providing gift samples of paracetamol; Uni Drugs Innovative PharmaTechnologies Ltd., Indore, India, for chlorzoxazone; and Aristo Pharma Ltd.,Mandideep, India, for aceclofenac. One of the authors is thankful to SureshPrajapati for technical assistance.

Address correspondence to R. Sharma, School of Pharmacy, Devi AhilyaVishwavidyalaya, Takshshila Campus, Khandwa Road, Indore 452001, M.P.,India. E-mail: rbsm73@yahoo.co.in

Analytical Letters, 41: 3297–3308, 2008Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print=1532-236X onlineDOI: 10.1080/00032710802515086

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were linear over the concentration ranges of 5–50 mg=ml for acetaminophen andchlorzoxazone, and 5–30 mg=ml for aceclofenac. All the validation study wasfound statistically significant because all the statistical parameters were withinthe acceptance range (i.e., COV %< 2.0 and S.D.< 1.0 for both accuracy and pre-cision). The limit of detection (LOD) values were 16.2, 14.6, and 4.8 ng=ml, andLOQ values were 49.0, 46.5, and 14.5 ng=ml for acetaminophen, chlorzoxazone,and aceclofenac respectively. High recovery and low COV % revealed the reliabilityof the method for quantitative study of three drugs in Micronac-MR tablets. Themethod is a rapid and cost-effective quality-control tool for routine quantitativeanalysis of acetaminophen, chlorzoxazone, and aceclofenac in tablet dosage form.

Keywords: Aceclofenac, acetaminophen, chlorzoxazone, ICH guidelines,RP-HPLC

INTRODUCTION

A fixed-dose combination tablet containing acetaminophen, chlorzoxazone,and aceclofenac is clinically used for the management of pain and muscu-loskeletal spasm along with inflammation. Chemically, acetaminophen is4-hydroxy acetanilide, used as an analgesic and antipyretic drug; chlor-zoxazone is 5-choro-2-benzoxazolol, a commonly prescribed musclerelaxant; and aceclofenac-2[2,6-dichlorophenyl) amino] benzoic acid car-boxymethyl ester is an analgesic and nonsteroidal anti-inflammatorydrug. Acetaminophen is official in BP and IP (British Pharmacopoeia2002; Indian Pharmacopoeia 1996), chlorzoxazone in USP (United StatesPharmacopoeia 2006), and aceclofenac in BP (British Pharmacopoeia2002). IP and BP suggest titrimetric and ultraviolet (UV) spectrophoto-metric assay method for acetaminophen in bulk and tablet formulations.BP suggests a potentiometric assay method for aceclofenac in bulk drugs.

A literature survey revealed that high-performance liquid chromato-graphic (HPLC; Hinz et al. 2003), densitometric (El-Saharty, Refaat, andel-Khateeb 2002), spectrofluorimetricm (El-Kousy 1999), and colori-metric (Zawilla et al. 2002) methods have been reported for the estima-tion of aceclofenac in pharmaceutical dosage forms. Also simultaneousestimation of acetaminophen, chlorzoxazone, and aceclofenac in tabletsby UV spectrophotometry (Garg, Saraf, and Saraf 2007) has beenreported. But so far, no chromatographic method has been reportedfor simultaneous estimation of acetaminophen, chlorzoxazone, and ace-clofenac in combined dosage form; hence it is essential to develop a chro-matographic method for simultaneous estimation of three drugs in tabletformulation. As HPLC methods were widely used for routine analysis ofdrugs because of its sensitivity and accuracy, so in the present work anew, simple, and specific RP-HPLC method was developed for

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simultaneous estimation of acetaminophen, chlorzoxazone, and aceclofenacin tablet dosage form.

EXPERIMENTAL

Materials

A pure sample of acetaminophen was obtained from Zest Pharma, chlor-zoxazone from Uni Drugs Innovative Pharma Technologies Ltd., andaceclofenac from Aristo Pharma Ltd. The commercially available tablets,Micronac-MR (label claim: acetaminophen, 500 mg; chlorzoxazone,250 mg; and aceclofenac, 100 mg), was procured from a local market.All the chemical and reagents used were of HPLC grade and purchasedfrom Spectrochem Pvt. Ltd. HPLC-grade water was obtained using themillipore water purification system.

Chromatographic System and Conditions

Analysis was performed with a Shimadzu chromatograph equipped withan LC-10 AT vp solvent-delivery system with universal loop injector(Rheodyne 7725 i) with injection capacity of 20 mL. Detector consistedof photodiode array detector SPD-10 AVP UV-visible (UV-vis) detector.Separation was carried out on a Phenomenex Luna C18 instrument(5 mm� 25 cm� 4.6 mm i.d.) under reversed-phase partition chromato-graphic conditions. The equipment was controlled by a PC with the chro-matography software. The work was carried out in an air-conditionedroom maintained at temperature 25� 2�C. The mobile phase, a mixtureof buffer (in 500 ml of water, add 1 ml of triethylamine), methanol, andacetonitrile in the ratio 215:130:155, was prepared, with pH adjusted to6.5 using dilute orthophosphoric acid. The flow rate was 1.5 ml=min,and the analytes were monitored at 275 nm. The total run time was10 min. Before analysis, both the mobile phase and sample solutions weredegassed by the use of a sonicator and filtered through 0.2-mm filterpaper. The identities of three compounds were established by comparingretention time of the sample solution with those of standard solutions.

PROCEDURES

Preparation of Standard Solution and Construction of Calibration Plots

The standard stock solutions of acetaminophen, chlorzoxazone, and ace-clofenac were prepared by dissolving 50 mg of each drug in 100 ml of

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mobile phase. From this solution, 10 ml of solution were taken anddiluted to 50 ml with the same to get a solution containing 100 mg=mlof each drug. From the stock solutions, further dilutions were preparedby diluting required volume of solution with mobile phase, and their areawas noted by injecting 20 ml into the system. After that, a calibrationcurve was plotted between concentration against their respective areafor acetaminophen, chlorzoxazone, and aceclofenac separately. Fromthe calibration curve, it was found that acetaminophen and chlorzoxa-zone had linearity ranges between 5 and 50 mg=ml, whereas aceclofenachad a range between 5 and 30 mg=ml.

Assay of Tablet Formulation

For analysis of the tablet dosage form, 20 tablets were weighted individu-ally and their average weight was determined. Then they were crushed tofine powders, and a powder equivalent to the weight of 50 mg of acetami-nophen was transferred to 100-ml volumetric flask and dissolved inmobile phase. The solution was shaking vigorously for 15 min and fil-tered through Whatman no. 41 filter paper, and the residue was washedwith mobile phase. Then volume was made up to the mark with mobilephase. From this solution, 10 ml of solution was taken and diluted to50 ml with the same to get a solution containing 100 mg=ml of acetamino-phen and corresponding concentrations of chlorzoxazone and aceclofe-nac. The solution contained acetaminophen, chlorzoxazone, andaceclofenac in the proportions of 10:5:2.

From this solution, 3.0 ml of solution was transferred in 10-ml volu-metric flask and diluted with the same to obtain a final concentration of30 mg=ml of acetaminophen, 15 mg=ml of chlorzoxazone, and 6 mg=ml ofaceclofenac. The amounts of acetaminophen, chlorzoxazone, and aceclo-fenac per tablet were calculated by extrapolating the value of area fromthe calibration curve. Analysis procedure was repeated six times withtablet formulation. The result of analysis of tablet formulation isreported in Table 1.

RESULTS AND DISCUSSION

HPLC Method Development and Optimization

Column chemistry, solvent type, solvent strength (volume fraction oforganic solvent(s) in the mobile phase and pH of the buffer solution),detection wavelength, and flow rate were varied to determine the

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chromatographic conditions giving the best separation. The mobile-phaseconditions were optimized so that the solvent and excipients did notinterfere with the components. Other criteria such as time required foranalysis, appropriate range for eluted peaks, assay sensitivity, solventnoise, and use of the same solvent system for extraction of drug from for-mulation matrices during drug analysis were also considered.

After trying different columns, the final choice of the stationary phasethat gave a satisfactory resolution and run time was the reversed-phasecolumn Luna C18 (5 mm� 25 cm� 4.6 mm i.d.). A series of aqueousmobile phases containing buffer solutions of different pH values in com-bination and different volume fractions of methanol and acetonitrile asmodifiers were also tested. The best results were obtained by use of a mix-ture of buffer (500 ml of water and 1 ml triethylamine), methanol, andacetonitrile in the ratio 215:130:155, with pH adjusted to 6.5 using diluteorthophosphoric acid. The flow rate was determined by testing the effectof different flow rate on the peak area and resolution; a flow rate of1.5 ml=min found to be optimum. All experiments were carried out atambient temperature.

To determine the appropriate wavelength for simultaneous determi-nation of acetaminophen, chlorzoxazone, and aceclofenac, solutions ofthese compounds in the mobile phase were scanned by a UV-vis spectro-photometer (Shimadzu 1700) in the range 200–400 nm. From the overlaidUV spectra, suitable wavelength considered for monitoring the drugs was275 nm. Solutions of each substance in the mobile phase were alsoinjected directly for HPLC analysis, and the responses (peak area) wererecorded at 275 nm. It was observed there was no interference from themobile phase or baseline disturbance, and these three drugs absorbed wellat 275 nm. It was, therefore, concluded that 275 nm is the most appropri-ate wavelength for analysis of both the drugs with suitable sensitivity.

Table 1. Result of intraday and interday precision, LOD and LOQ study

Values

Parameters ACT CHL ACL

LODa (ng=ml) 16.2 14.6 4.8LOQa (ng=ml) 49.0 46.5 14.5Intradaya (precision) (% COV) 0.9424 0.6495 0.5172Interday (precision) (% COV), n¼ 3 0.5186 0.5190 0.4327

Notes. ACT: acetaminophen, CHL: chlorzoxazone, ACL: aceclofenac, COV:coefficient of variation.

aAverage of six determinations.

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Under the optimum chromatographic conditions, the retentiontimes obtained for acetaminophen, chlorzoxazone, and aceclofenacwere 2.055, 5.096, and 7.605 min respectively (Figs. 1, 2, and 3). Theresolutions (Rs) between the three curves were 15.33 and 7.06. Theresult of capacity factor, tailing factor, and theoretical plate numberare reported in Table 2.

The values obtained for these properties (1< k< 10, Rs> 2) showsthese chromatographic conditions are appropriate for separation andquantification of both compounds. The number of plates (N) is a mea-sure of column efficiency, which shows the good separation efficiencyof the column used.

Figure 1. Chromatogram of acetaminophen and its retention time.

Table 2. Result of assay of tablet formulation

DrugLabel claim

(mg=tab)Amount

founda (mg=tab)Label

claim (%) SDa COV (%) SEa

ACT 500 499.45 99.89 0.2607 0.2609 0.1064CHL 250 249.57 99.83 0.4158 0.4165 0.1697ACL 100 99.87 99.87 0.4839 0.4845 0.1975

Notes. ACT: acetaminophen, CHL: chlorzoxazone, ACL: aceclofenac, SD:standard deviation, COV: coefficient of variation, SE: standard error.

aAverage of six estimation of tablet formulation.

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Validation of the Developed Method

The method was validated for linearity, accuracy, precision, repeatability,selectivity, and specificity study. All the validation study was carried outby replicate injection of the sample and standard solutions.

Linearity

The linearity was determined for three drugs, acetaminophen, chlorzox-azone, and aceclofenac, separately by plotting a calibration graph of peakarea against their respective concentration. From the calibration curve, itwas clear that acetaminophen and chlorzoxazone had linearity between 5and 50 mg=ml, whereas aceclofenac had a range between 5 and 30 mg=ml.The linear regression equation for three drugs was

Acetaminophen: y¼ 12648x� 2149.3 (r2¼ 0.9975)Chlorzoxazone: y¼ 18782xþ 6039.5 (r2¼ 0.9982)Aceclofenac: y¼ 23223x� 13906 (r2¼ 0.9965)

where y is peak area and x is concentration.

Accuracy

Accuracy of the developed method was conformed by doing a recoverystudy as per ICH norms at three different concentration levels (80%,100%, 120%) by replicate analysis (n¼ 3). Standard drug solutions wereadded to a preanalyzed sample solution, and then percentage of drugcontent was calculated. The result of the accuracy study are reported inTable 3.

Table 3. Result from system-suitability study

Property Acetaminophen Chlorzoxazone Aceclofenac

Rt 2.055 5.096 7.605Tf 1.23 1.31 0.99k0 2.44 2.45 2.34N 3491 6983 4214Rs 15.33 7.06 —

Notes. Rt: retentiontime, Tf: tailing factor, k0: capacity fac-tor, N: theoretical plate number, Rs: resolution.

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From the recovery study, it was clear that the method is very accu-rate for quantitative estimation of acetaminophen, chlorzoxazone, andaceclofenac in tablet dosage form because all the statistical results werewithin the acceptance range (i.e., COV %< 2.0 and SD< 1.0).

Precision and LOD and LOQ

Precision was determined by studying the repeatability and intermediateprecision. Repeatability result indicates the precision under the sameoperating conditions over a short interval time and interassay precision.The standard deviation, coefficient of variance, and standard error werecalculated for three drugs. The results are mentioned in Table 1. Inter-mediate precision was carried out by doing intra- and interday precisionstudies. In the intraday study, the concentrations of three drugs were cal-culated on the same day at an interval of 1 h. In the interday study, theconcentrations of drug contents were calculated on three different days,and the study expresses within-laboratory variation in different days.In both intra and interday precision studies for the methods, COV%values were not more than 2.0%, which indicates good intermediateprecision (Table 4).

LOD and LOQ studies were carried out to evaluate the detection andquantization limits of the method to determine the presence of anyimpuritiesby using following equation:

LOD ¼ 3:3 r=S

LOQ ¼ 10 r=S

where r is the standard deviation and S is the slope of the curve.The result is reported in Table 4. The developed method was precise

for quantitative study because the precision study was found statisticallysignificant (COV %< 2.0 and SD< 1.0 for intra- and interday studies).

Selectivity and Specificity

To check the selectivity of the developed method, solutions of the threedrugs were injected into the system, and three sharp peaks for acetamino-phen, chlorzoxazone, and aceclofenac were obtained at retention times of2.055, 5.096, and 7.605 min respectively in reference to placebo solution.Specificity of the method was assessed by comparing the chromatogramsobtained from standard drugs (Figs. 1, 2, and 3) with the chromatogramobtained from tablet (Fig. 4) solutions. Because the retention time of

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standard drugs and the retention time of three drugs in sample solutionswere the same, the method was specific. The developed method wasspecific and selective as no interference of excipients was found.

Figure 3. Chromatogram of aceclofenac and its retention time.

Figure 2. Chromatogram of chlorzoxazone and its retention time.

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CONCLUSION

A new, reversed-phase HPLC method has been developed for simulta-neous quantization of acetaminophen, chlorzoxazone, and aceclofenacin tablet formulation. It has been shown that the developed methodachieved accuracy, reproducibility, repeatability, linearity, precision,and selectivity, which prove the reliability of the method. The run timeis relatively short, 10 min, which enables rapid quantization of many sam-ples in routine and quality-control analysis of tablet formulation. Thesame solvent was used throughout the experimental work, and no inter-ference of any excipients matrices was found. The result shows that themethod could find practical application as a quality-control tool forthe simultaneous estimation of three drugs from their combined dosageform in a quality-control laboratory.

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El-Kousy, N.M. 1999. Spectrophotometric and spetrofluorimetric determinationof etodolac and aceclofenac. J. Pharm. Biomed. Anal., 20: 185–188.

Figure 4. Chromatogram of acetaminophen, chlorzoxazone, and aceclofenac insample solution and their retention times.

Estimation of Acetaminophen, Chlorzoxazone, and Aceclofenac 3307

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Garg, G., Saraf, S., and Saraf, S. 2007. Simultaneous estimation of aceclofenac,paracetamol and chlorzoxazone in tablets. Indian J. Pharm. Sci., 69(5):692–694.

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United States Pharmacopoeia. 2006. United States Pharmocopeial Convention,Inc., Rockville, MD, USA, 500–501.

Zawilla, N.H., Mohammad, M.A., El-Kousy, N.M., and El-Moghazy, A.S. 2002.Determination of aceclofenac in bulk and pharmaceutical formulation.J. Pharm. Biomed. Anal., 27: 243–246.

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