Development and validation of a stability-indicating HPTLC method ...

Research articleInt J Pharm Biomed Sci 2011, 2(2), 55-60ISSN No: 0976-5263Research DropsPharmaInterScience PublishersDevelopment and validation of a stability-indicating HPTLC methodand HPLC method for simultaneous estimation of lornoxicam andparacetamol in combined tablet dosage formsDinesh Kumar Jain*,Nitin Dubey, Yash SharmaCollege of Pharmacy, IPS Academy,Indore (Madhya Pradesh)-462 046, India*Correspondence:Mr. Dinesh Kumar JainPhone: +91 0731 4041627E-mail: yashmaan2007@rediffmail.comA simple, precise and stability-indicating high-performance liquidchromatography (HPLC) and high-performance thin-layer chromatographic(HPTLC) method for simultaneous estimation of lornoxicam (LOR) andparacetamol (PCM) in tablet formulations has been developed and validated. TheHPLC separation was achieved on a Phenomenex Luna C18 column usingmethanol: acetonitrile: water (50:30:20, v/v/v) as mobile phase (pH 3.8) at a flowrate of 1.0 mL/min at ambient temperature. Quantitation was achieved with UVdetection at 290 nm over the concentration range 2-10 μg/mL and 5-100 μg/mLwith mean recovery of 99.71 ± 0.189 % and 99.55 ± 1.10% for LOR and PCM,respectively. LOR (290 nm) and PCM (256 nm) were chromatographed on silicaGel 60 F254 TLC plate using chloroform: methanol: ethyl acetate: acetic acid(20:15:55:10, % v/v/v/v) as the mobile phase and then scanned. The compact bandsat Rf 0.23 ± 0.01 and 0.60 ± 0.02 respectively, were observed for LOR and PCM.Linear regression analysis revealed linearity in the range of 300 – 550 ng/band and100-500 ng/band for LOR and PCM respectively. Drugs were subjected to acid andalkali hydrolysis, oxidation, thermal and photo-degradation treatments. Thedegraded products were well separated from the pure drug. Statistical analysisproved that the method is precise, accurate, selective and economical and may beused for routine simultaneous estimation of LOR and PCM for tablet formulations.Key words: Lornoxicam, Paracetamol, Simultaneous estimation, HPLC methoddevelopment, HPTLC method development, Method validation, Stability-indicatingmethodReceived: 04 Jun 2011 / Revised: 15 Jun 2011 / Accepted: 17 Jun 2011 / Online publication: 28 Jun 20111. INTRODUCTIONLornoxicam (LOR) is chemically known as 6-chloro-3-[hydroxy(pyridin-2- ylamino)methylene]-2-methyl-2,3-dihydro-4H-thieno[2,3-e][1,2]thiazin-4-one 1,1-dioxidebelongs to the class of cyclooxygenase inhibitor of nonsteroidalantiinflammatory drug (NSAID) of the oxicam classwith analgesic, antiinflammatory and antipyretic properties.Paracetamol (PCM) is chemically known as N-(4-hydroxyphenyl) acetamide. PCM inhibits prostaglandinbiosynthesis by blocking the enzyme cyclooxygenase [1-4].The chemical structures of LOR and PCM are shown inFig.1. Literature survey revealed that few HPLC and HPTLCmethods have been reported for the estimation ofParacetamol alone or in combination with other drugs frompharmaceutical formulation and biological fluids [5].a)b)Fig.1. Chemical structures of a) Lornoxicam and b) Paracetamol©2011 PharmaInterScience Publishers. All rights reserved. www.pharmainterscience.com

Dinesh Kumar Jain et al., Int J Pharm Biomed Sci 2011, 2(2), 55-6056Several HPLC, UV spectrophotometric and otherchromatographic methods have been reported fordetermination of Lornoxicam from pharmaceuticalformulation and biological fluids [6]. But none of thesemethods demonstrate the simultaneous determination of thesetwo drugs in tablets dosage from.This paper describes simple, accurate, precise, andsensitive reversed-phase RP-HPLC and HPTLC methods forthe simultaneous estimation of LOR and PCM in a combinedtablet dosage form.2. MATERIALS AND METHODS2.1 MaterialsLornoxicam and paracetamol were obtained fromGlenmark pharmaceutical ltd., Mumbai, as a gratis sample,respectively. HPLC grade chemicals (acetonitrile, methanoland water) were obtained from E. Merck (Mumbai, India)The tablet dosage form (Label claim: 8 mg Lornoxicam, and500 mg Paracetamol) was procured from the local market andother reagents such as chloroform, acetonitrile, methanol,ethyl acetate and ammonia were obtained from E. Merck(Mumbai, India) which were of analytical grade.2.2 Apparatus and chromatographic conditions2.2.1 HPLC methodThe mobile phase consist of acetonitrile: methanol: water(50:30:20, %v/v/v) at a flow rate of 1.0 mL/min. Before use,the mobile phase was degassed by an ultrasonic bath andfiltered using 0.4 μm membrane filter before use. Separationwas performed at room temperature on HPLC system havinga pump (Shimadzu LC 10ATVP ) with 20 µL Rheodyneinjector , Phenomenex Luna C18 (5 µm x 25cm x 4.6mm i.d)column and SPD-10 AVP photodiode array (PDA) UV-Visible detector set at 290 nm and equipped with CLASS-VPsoftware (Shimadzu. Kyoto, Japan).2.2.2 HPTLC methodChromatography was performed over precoated silica gelaluminum plates 60 F254 (20 × 10 cm, 200 μm thickness; E.Merck, Germany) as stationary phase. Samples were spottedin the form of distinct bands (6 millimeter; 10 mm apart) witha CAMAG 100 μL syringe using a Linomat V (CAMAG,Switzerland) sample applicator. Equipment parameters wereoptimized for smooth working (scanning speed 100 nm/sec;slit dimension 6.00 x 0.45 mm). Linear ascendingdevelopment was carried out in a 20 cm × 10 cm twin troughglass chamber (CAMAG, Switzerland) previously saturatedwith optimized mobile phase for 20 min at room temperature(30ºC) and relative humidity (RH) of 55 ± 5%. The plateswere developed to the distance of 10 cm and dried using hairdryer. The mobile phase was chloroform: methanol:ethylacetate: ammonia (2.0:1.5:5.5:1, %v/vv/v) withdensitometric analysis at 290 nm in absorbance mode withCAMAG TLC scanner III and winCATS software (Ver.1.2.0).2.3 Method validation2.3.1 LinearityFor HPLC analysis, the calibration graph was plotted over6 different concentrations in the range of 2–10 μg/mL and 5-100 μg/mL for LOR and PCM. Accurately measured workingstandard solution aliquots of LOR and PCM were transferredto a series of 10 mL volumetric flasks and diluted to the markwith mobile phase. Aliquots (20 μL) of each solution wereinjected chromatographed in six replicates.For HPTLC analysis, a stock solution containing 1000ng/μL each for LOR and PCM were prepared in methanol.Different volumes of this solution were applied to the plateresulting in application of 300-550 ng/band for LOR and100-500 ng/band for PCM, respectively (n=6). Peak areaswere plotted against corresponding concentrations to furnishthe calibration plot.2.3.2 PrecisionRepeatability of sample application and measurement ofpeak areas were assessed by chromatography of six replicatesof the same concentration (300 ng/band for LOR and 100ng/band for PCM). Intra-day and inter-day precision fordetermination of LOR and PCM were measured at threedifferent concentration (300, 350, 400ng/band) and (100,200, 300ng/band). The intraday and Interday precisions of theproposed methods were determined by analyzing standardsolution of LOR and PCM at three different concentrations(4.0, 8.0, and 12.0 μg/mL for the HPLC method and 200,300, and 500 ng/band for the HPTLC method) three times onthe same day and on three different days.2.3.3 RobustnessThe robustness of the method was determined byintroducing small changes in certain chromatographicparameters, such as changing the composition and the pH ofmobile phase in the range ±5%.2.3.4 SpecificityThe specificity of the method was determined by analysisof drug standards and samples. The band for LOR and PCMin the sample was identified by comparing the RF value andspectrum of the band with those of the band from a standard.The peak purity of LOR and PCM were assessed bycomparing spectra acquired at three different positions on thepeak, i.e. the peak start, peak apex, and peak end positions ofthe band [7, 8].©2011 PharmaInterScience Publishers. All rights reserved. www.pharmainterscience.com

Dinesh Kumar Jain et al., Int J Pharm Biomed Sci 2011, 2(2), 55-60Table 1System suitability parameter for HPLC methodParameters LOR PCMCalibration range (µg/mL) 2-40 8-150Theoretical plate number 4009 2311HETP a 0.0073 0.004Tailing factor 1.52 1.86Capacity factor (k’) 0 1.78Resolution - 6.58a HETP = Height equivalent to theoretical plate, cmTable 2Summary of validation dataS. No. Validation parameters Observation of HPTLC methodLORPCM1 Linearity range300-550 100-500(ng/band)2 Slope 10.48 4.663 Intercept 1226.7 -137.044 Wavelength (nm) 249 3705 Correlation coefficient 0.993 0.9966 Accuracy (n=5) 100.39±1.42 100.56±1.27 Intraday Precision (n=3) 1971.7±24.85 1356.6±23.378 Interday Precision (n=3) 5376.6±88.37 1361.6±35.549 Repeatability (n=6) 3814.8±66.61 313.88±6.3410 Recovery (n=3) 98.91±1.39 98.84±0.5711 LOD (ng/band) 0.62±0.02 0.92±0.0312 LOQ (ng/band) 1.26±0.01 2.17±0.0213 Robustness Robust Robust14 Specificity Specific Specific2.3.5 Limit of detection (LOD) and Limit of quantitation(LOQ)The LOD with signal-to-noise (S/N) ratio of 3:1 and theLOQ with S/N ratio of 10:1 were calculated for LOR andPCM using the following equations LOD = 3.3 σ/S, LOQ =10 σ/S where σ is the standard deviation of the response andS is the standard deviation of the y-intercept of the regressionline for HPLC analysis. For HPTLC analysis the limit ofdetection (LOD) and the limit of quantification (LOQ) wasdetermined by diluting known concentration of standardstock solution until the average responses wereapproximately 3 or 10 times the responses of the blank.2.3.6 RecoveryTo check the recovery of the drug at different levels informulations, samples were spiked with 50, 100, and 150 %of LOR and PCM standards and the mixtures were analyzedin triplicate by the proposed method.2.4 Stability of Sample SolutionsSolutions at two different concentrations (300 and350ng/band for LOR and 200 and 300ng/band for PCM)were prepared from stock solution and stored at roomtemperature for 24, 4.0, 2.0 1.0, 0.5 and 0.2h. They were thenchromatographed on the same plate. The chromatogramswere evaluated for additional spots if any.2.5 Assay of the marketed formulationTwenty tablets were weighed and crushed. An amount ofpowder equivalent to 100 mg PCM was transferred to a 100mL volumetric flask and extracted with methanol for 30 minby shaking mechanically and volume was adjusted to markwith the same solvent. 1mL of this solution was diluted to 10mL with methanol. An appropriate volume of 5 μL wasassayed by the chromatographic procedure described above.No interferences were observed from the excipientscommonly present in the tablets.2.6 Forced degradation studiesA stock solution containing 10 mg for each (LOR andPCM) in 10 mL methanol was prepared. This solution wasused for forced degradation to provide an indication of thestability-indicating property and specificity of the method.The average peak area of sex replicates of LOR and PCM(1000 ng/band) was used for analysis.2.6.1 Acid degradationMethanolic solution of each drug (10 mg) was separatelydissolved in 10 mL of 1M HCl and these solutions were keptfor 8 h at room temperature in dark in order to exclude thepossible degradative effect of light. The solutions (1 mL)were taken and neutralized and then diluted up to 10 mL withmethanol. The resultant solutions were applied on TLC platein triplicate (10 μL each, i.e. 1000 ng/band). The plate waschromatographed as described above.2.6.2 Base degradationMethanolic solution of each drug (10 mg) was separatelydissolved in 10 mL of 1 M NaOH solution. These solutionswere kept for 8 h at room temperature in dark in order toexclude the possible degradative effect of light. The solutions(1 mL) were taken and neutralized and then diluted up to 10mL with methanol. The resultant solutions were applied onTLC plate in triplicate (10 μL each, i.e. 1000 ng/band). Theplate was chromatographed as described above.2.6.3 Oxidative degradationEach drug (10 mg) was dissolved in 10 mL of methanolicsolution of hydrogen peroxide (10% v/v) and kept for 8 h atroom temperature in the dark, to exclude the possibledegradative effect of light. The solution (1 mL) was thendiluted to 10 mL with methanol and treated as described foracid and base-induced degradation.57©2011 PharmaInterScience Publishers. All rights reserved. www.pharmainterscience.com

Dinesh Kumar Jain et al., Int J Pharm Biomed Sci 2011, 2(2), 55-6058Fig. 2.HPLC chromatogram of LOR and PCMFig.3. (a) HPTLC chromatogram of LOR and PCM from a tablet formulation showing LOR (300 ng/band, R f = 0.23±0.01) and PCM (400ng/band,R f =0.60±0.02) peaks (b) overlay spectra of LOR and PCM2.6.4 Photochemical degradationEach drug solution was left in sunlight for 8h. Theresultant solution was treated as described for hydrogenperoxide-induced degradation.2.6.5 Dry heat degradationThe powdered drug was stored for 3h. under dry heatconditions at 55°C. A solution of the treated powder was thenprepared for each drug and 1000 ng/band was applied to aplate in triplicate. The plate was then chromatographed andtreated as described above.3. RESULTS AND DISCUSSIONTo optimize the HPLC parameters, several mobile phasecompositions were tried. A satisfactory separation of LORand PCM with good peak symmetry and steady baseline wasobtained with the mobile phase methanol: acetonitrile: water(50:30:20, %v/v/v) (pH 3.8) at a flow rate of 1.0 mL/min.Complete resolution of the peak with clear baseline©2011 PharmaInterScience Publishers. All rights reserved. www.pharmainterscience.com

Dinesh Kumar Jain et al., Int J Pharm Biomed Sci 2011, 2(2), 55-6059Table 3Assay result of marketed formulationS. No. Actual concentration (mg) Concentration found (mg) % ContentLOR PCM LOR PCM LOR PCM1 300 400 302.43±4.22 400.31±1.64 101.02±1.34 100.08±1.92Table 4Forced degradation studiesSample exposure conditions LOR PCMNumber of degradation products % Recovery (n=3) Number of degradation products % Recovery (n=3)(R f value)(R f value)1 M HCl,8h, RT 3 (0.28,0.72, 0.88) 89.23 ±2.17 2 (0.75,0.88) 96.02 ±3.541 M NaOH, 8h, RT 4 (0.01,0.04, 0.72,0.88) 79.0 ±2.94 1(0.4) 76.05 ±1.3710%,H 2 O 2 , 8h, RT 2 (0.71,0.87) 74.34 ±7.15 2(0.40,0.64) 75.14 ±0.82Sunlight, 8h 1(0.76) 45.2 ±1.91 1 (0.79) 13.53 ±1.31Heat, 3h, 55°C 2 (0.31,0.75) 99.67 ±2.43 2 (0.76,0.96) 93.75 ±0.84a) b)Fig.4. HPTLC chromatogram obtained from forced degradation studies (a) acid degradation (1 M HCl, 8h, RT) showing 3 degradants at R f values 0.28, 0.72,0.88 for LOR and 2 degradants at R f values 0.75, 0.88 for PCM and (b) base degradation (1 M NaOH, 8h, RT) showing 4 degradants at R f values 0.01, 0.04,0.72, 0.88 for LOR and 1 degradant at R f value 0.4 for PCMseparation was obtained at 290 nm (Fig.2). The systemsuitability test parameters are shown in Table 1.In the development of HPTLC method, the mixture ofchloroform:methanol:ethyl acetate:ammonia (20:15:55:10, %v/v/v/v) was selected as a mobile phase as it could resolveLOR and PCM spots with a better peak shape and offered anoptimum separation at R f 0.23 ± 0.01 and 0.60 ± 0.02 (n=6)for LOR and PCM respectively (Fig.3a).Linear correlation was obtained in the range of 2-10μg/mL and 5-100 μg/mL for the HPLC method and 300-550ng/band and 100-500 ng/band for HPTLC method for LORand PCM, respectively. The average linear regressionequation was represented as Y = 10.348X + 1226.7 for LOR,and Y = 4.665X + 137.04 for PCM, where X is theconcentration of drug and Y is the peak area. The overlayspectra of LOR, PCM are shown in Fig.3b. The repeatabilityof the method was checked by spotting 4 μL of a combinedstandard solution six times on the plate (n= 6); the RSD forthe peak area was found to be 0.72 and 1.2 for LOR andPCM using HPLC and 1.24 and 1.02 for LOR and PCM forHPTLC. The intra-day and inter-day The RSD values werefound to be

Dinesh Kumar Jain et al., Int J Pharm Biomed Sci 2011, 2(2), 55-60ng /band for LOR and PCM respectively. Different validationparameters for the proposed HPTLC method are summarizedin Table 2.There were no indications of compound instability insample solutions containing concentrations 400 and 600ng/band for LOR and 100 and 200 ng/band for PCM storedfor up to 24 h.Six replicate determinations were performed on thecommercially available tablet. For LOR and PCM recoverywas found to be 99.75±0.0642% and 99.71±0.189%,respectively for HPLC and for HPTLC the spots of Rf 0.23and 0.60 were observed in chromatograms obtained fromdrug samples extracted from tablets. There was Nointerference was observed from the excipients commonlypresent in the tablets. It may, therefore, be inferred thatdegradation of LOR and PCM had not occurred in themarketed formulations analysed by this method. The lowRSD indicated that the method is suitable for routinesimultaneous estimation of LOR and PCM in pharmaceuticaldosage forms. The content and percentage of LOR and PCMin market samples are presented in Table 3.Drug recovery at the level of 89.23 % and 96.02 % forLOR and PCM respectively, from the acid-stressed samples(Fig.4), indicated significant degradation in acidic condition(Table 4). Drug recovery from base- stressed sample 79.0 %and 76.05 % for LOR and PCM respectively (Table 4). Thedifferent R f value obtained from acid and base induceddegradation product indicates that the acid degradationproducts are different from alkaline degradation product. Thechromatogram treated with 10 % (v/v) H 2 O 2 revealed peaksfor LOR and PCM (Table 4). Drug recoveries were found tobe 74.34 % and 75.14 % for LOR and PCM respectively.Drug recovery was found to be 45.2 % and 13.53 % for LORand PCM respectively, from samples of the photochemicaldegradation study, suggestion significant degradation(Table4). Significant degradation was also evident in dry heatsamples. Drug recovery was found to be at the level of 99.67% and 93.75 % for LOR and PCM respectively [9, 11].4. CONCLUSIONSThe proposed HPTLC and HPLC methods were found tobe simple, fast, accurate, precise, and sensitive. Thus, it maybe used for routine analysis of LOR and PCM in combinedtablet dosage form.ACKNOWLEDGMENTSThe authors wish to express their gratitude to Glen markpharmaceutical ltd., Mumbai and for gratis samples ofLornoxicam and Paracetamol. The authors are thankful toDirector, SICART, Vallabh Vidyanagar, Gujarat foranalytical facilities.REFERENCES[1] Altun ML. HPLC method for the analysis of paracetamol, caffeine anddipyrone. Turk J Chem 2002, 26, 521-528.[2] Akay C, Degim T, Sayal A, Aydin A, Ozakan Y, Gul H. Rapid andsimultaneous determination of acetylsalicylic acid, paracetamol andtheir degradation and toxic impurity products by hplc in pharmaceuticaldosage forms. Turk J Med Sci 2008, 38, 167–173.[3] Shirkhedkar AA, Shaikh AM, Surana SJ. Simultaneous determination ofparacetamol and piroxicam in tablets by thin layer chromatographycombined with densitometry. Eurasian J Anal Chem 2008, 2, 258-267.[4] Kwiecien A, Krzek J, Biniek L. TLC determination of azithromycin inpharmaceutical preparations. J Planar Chromatogr 2008, 21, 177-181.[5] Bhavsar SM, Patel DM, Khandhar AP, Patel CN. Validated RP-HPLCmethod for simultaneous estimation of lornoxicam and thiocolchicosidein solid dosage form. J Chem Pharm Res 2010, 2, 563-572.[6] Cetin, I., Kocak, N., Aycan, S., C.B.U. J Sci 2009, 11, 18-23.[7] Bipin HM, Sachin BM. HPTLC densitometric analysis of candesartancilexetil and hydrochlorthiazide in tablets. J Planar Chrom Mod TLC2008, 21, 173-176.[8] Dubey, N., Dubey, N., Mehta, R.S., Saluja, A.K., J AOAC Int 2009,92(6), 1617.[9] Dubey, N., Dubey, N., Mehta, R.S., Saluja, A.K., J AOAC Int 2009,92(4), 1021.[10] Dubey N, Dubey N, Mehta RS, Saluja AK. J AOAC Int 2009, 92, 779.[11] Shethi PD. HPTLC High Performance Thin Layer Chromatography,CBS Publishers, New Delhi 1996.[12] Beckett AH. Practical pharmaceutical chemistry, CBS Publication 1997.[13] Reynolds JEF, Parfitt K, Partons AV, Sweetman SC. The ExtraPharmacopoeia 1996, Martindale, London, 82.60©2011 PharmaInterScience Publishers. All rights reserved. www.pharmainterscience.com