Single Rp-hplc Method for the Quantification of Aceclofenac, Paracetamol and Chlorozoxazone in...

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490

International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012 471

SINGLE RP-HPLC METHOD FOR THE QUANTIFICATION OF ACECLOFENAC, PARACETAMOL ANDCHLOROZOXAZONE IN FORMULATIONS

Hari kishan Reddy Ganthi1*, Hanimi Reddy Bapatu2, Maram Ravi Kumar3, Useni Reddy Mallu1,1Dept. of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India; 2Department of Chemistry,JNT University, Kukatpally, Hyderabad, AP, India-500072.; 3 AR&D, Custom Pharmaceutical Services,Dr. Reddy’s Laboratories Ltd, Bachupally, Hyd-72, India.

INTRODUCTION

INTRODUCTION

ISSN:2249-5347IJSID

International Journal of Science Innovations and Discoveries An International peerReview Journal for Science

Research Article Available online through www.ijsidonline.info

Received: 14-08-2012

Accepted: 17-10-2012

*Corresponding Author

Address:

Name:Hari Kishan Reddy GanthiPlace:Sri Krishnadevaraya UniversityAnantapur, AP, IndiaE-mail:[email protected]

ABSTRACT

Objectives: To develop a single RP-HPLC method for determination of Aceclofenac,Paracetamol and Chlorozoxazone contents in formulation.Methods: Chromatographic separation was achieved on Inertsil ODS 3V, 150 x4.6mm, 5µcolumn. Mobile phase composed of phosphate b u f f e r o f p H 6 . 0 a n da c e t o n i t r i l e i n t h e r a t i o n 6 7 : 3 3 v / v . 1.0ml per min flow rate and detectionwas at 275 nm.Results: High resolution was achieved with the simple mobile phase composition andretention time of Paracetamol, Chlorozoxazone, and Aceclofenac are about 2.1min, 8.8minand 20.7min, respectively. The area of all ingredient peaks were a linear function ofconcentration in the range 150.4 to 752.3 ppm for Paracetamol, 120.2 to 761.4 ppm forChlorozoxazone and 29.9 to 159.9 ppm for Aceclofenac and the correlation co-efficientvalue of all active ingredients within the limit (0.999).Conclusion: Proposed HPLC method was validated with specificity, linearity, accuracy,reproducibility and ruggedness and it is applicable for regular analysis.Keywords: Aceclofenac, Paracetamol, Chlorozoxazone and RP-HPLC method.



INTRODUCTION

Paracetamol (acetaminophen) is one of the most popular over-the-counter (OTC) analgesic and antipyretic drugs.Paracetamol (1-8) is available in different dosage forms: tablet, capsules, drops, elixirs, suspensions and suppositories.Aceclofenac is a non-steroidal anti-inflammatory drug (NSAID). It is used for the relief of pain and inflammation inrheumatoid arthritis, osteoarthritis and ankylosing spondylitis. The dose is 100 mg twice daily. It should not be given topeople with porphyria or breast-feeding mothers, and is not recommended for children.Chlorzoxazone is used to relieve pain and stiffness caused by muscle strains and sprains. It is used in combination withphysical therapy, analgesics (such as aspirin or acetaminophen), and rest. The side effects are upset stomach, drowsiness,dizziness, lightheadedness, weakness, skin rash or itching, yellowing of the skin or eyes and stomach pain.Chemical structures of all ingredients were represented in figure-1.All three ingredients are available in liquid pharmaceuticaldosage forms.

Paracetamol Chlorozoxazone Aceclofenac

Figure-1: Chemical structure of all ingredientsAll ingredients have reported methods for individual and other combination products, but the objective of the presentstudy is to develop a single RP-HPLC method for the estimation of Paracetamol, Aceclofenac and Chlorozoxazone informulations and the developed and validated method is simple, novel, rugged and linear.MATERIALS AND METHODS

Selection of mobile phase:Various buffer salts, pH values were tried with different organic solvents (acetonitrile or methanol) for the optimization ofmobile phase. Finally well shaped and high resolution was achieved with pH 6.0 phosphate buffer and acetonitrile at 67:33 v/vratio.Chemicals and reagents:Ortho phosphoric acid, triethyl amine (AR Grade) was procured from S.D fine chemicals. High pure (NLT 98.5%) standards(Aceclofenac, Paracetamol and Chlorozoxazone) were used for this study. HPLC grade acetonitrile were procured fromSpectrochem Pvt. Ltd. Water is prepared by mili Q system (Milli-pore).Buffer preparation: Diluted 2.0mL of ortho phosphoric acid to 1000 mL of water. Adjusted to pH 6.0 with triethyl amineFiltered through 0.45µm membrane filter and degassed.Mobile Phase: Mixed the buffer and acetonitrile in the ration 67:33 v/v.Diluent: Mobile phase.HPLC conditions:Column : Inertsil ODS 3V (150X4.6mm, 5μm)Wavelength : 275nmInjection volume: 20 μL



Flow rate : 1.0 mL/minTemperature : 30°CRun time : 30minMobile phase : Buffer and Acetonitrile 67:33 v/vPreparation of standard solution: For 100/ 500 / 500 mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration ofAceclofenac is about 2000µg/mL)Accurately weigh and transfer 100 mg of Paracetamol working standard, and 100 mg of Chlorzoxazone working standardin to 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute upto the mark with mobile phase and mix well. (Concentration of Aceclofenac is about 100 µg/mL, concentration ofParacetamol is about 500µg/mL and concentration of Chlorzoxazone is about 500µg/mL ).Preparation of standard solution: For 100/ 325/ 250 mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration ofAceclofenac is about 2000µg/mL)Accurately weigh and transfer 65 mg of Paracetamol working standard, and 50 mg of Chlorzoxazone working standard into 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up tothe mark with mobile phase and mix well. (Concentration of Aceclofenac is about 100 µg/mL, concentration ofParacetamol is about 325µg/mL and concentration of Chlorzoxazone is about 250µg/mL).Preparation of standard solution: For 100/ 500 mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration ofAceclofenac is about 2000µg/mL)Accurately weigh and transfer 100 mg of Paracetamol working standard, in to 200 mL volumetric flask, add to it about 10mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to the mark with mobile phase and mix well.(Concentration of Aceclofenac is about 100 µg/mL, concentration of Paracetamol is about 500µg/mL).Preparation of standard solution: For 100/ 500/15 mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration ofAceclofenac is about 2000µg/mL)Accurately weigh and transfer 100 mg of Paracetamol working standard, in to 200 mL volumetric flask, add to it about 10mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to the mark with mobile phase and mix well.(Concentration of Aceclofenac is about 100 µg/mL, concentration of Paracetamol is about 500µg/mL).Preparation of standard solution: For 100mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50.0 mL volumetric flask, add to itabout 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentrationof Aceclofenac is about 2000µg/mL)



Dilute the 10 mL of above Aceclofenac stock solution to 200 mL with mobile phase and mix well. (Concentration ofAceclofenac is about 100 µg/mL).Preparation of test solution: For 100/ 500 / 500 mg Tablets:Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weighaccurately the powder equivalent to 500 mg of Paracetamol into a 200 mL volumetric flask, add to it 20 ml ofacetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minuteswith intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix.Filter the solution through 0.45µ nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL withdiluent.Preparation of test solution: For 100/ 325 / 250 mg Tablets:Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weighaccurately the powder equivalent to 325 mg of Paracetamol into a 200 mL volumetric flask, add to it 20 ml ofacetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minuteswith intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix.Filter the solution through 0.45µ nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL withdiluent.Preparation of test solution: For 100/ 500 / 15 mg Tablets:Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weighaccurately the powder equivalent to 500 mg of Paracetamol into a 200 mL volumetric flask, add to it 20 ml ofacetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minuteswith intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix.Filter the solution through 0.45µ nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL withdiluent.Prepare the test solutions in duplicate.Procedure:Equilibrate the column with mobile phase for sufficient time until stable baseline is obtained. Inject blank, standard andsample preparation filter through 0.45µ nylon filter. Inject blank (diluent) in single, standard preparation in fivereplicates, and each test preparation into the chromatographic system and record the chromatograms. Inject standardpreparation as a bracketing after every six injections of test preparations. Evaluate the system suitability parameters fromthe standard chromatograms. The order of elution is Paracetamol, Chlorzoxazone and Aceclofenac.Calculation: % content= Test solution area x Std. Dilution factor x Std. PotencyStandard solution area x Test dilution factor

RESULTS AND DISCUSSION

System suitability:Standard solution was prepared as per the proposed test method and injected into the HPLC system. The results of thesystem suitability assessment for initial validation study parameters were tabulated in Table 1.



Table 1: System suitability

Sr.No. System Suitability parameter Observations LimitsAceclofenac Paracetamol Chlorzoxazone

1 The % RSD of peak area response forfive replicate injections of standard 0.3 0.1 0.2 NMT 2.02 Theoretical plates 2963 1506 3986 NLT 10003 Tailing factor 2.0 1.8 1.9 NMT 2.0

Precision Studies:

System Precision:Standard solution of working standard was prepared as per the proposed test procedure for repeatability studies. Fivereplicate injections were injected into the HPLC system. % RSD for the peak responses as the peak area was calculated,Results are tabulated in Table 2.

Table 2: System Precision

Injection No. Area ResponseAceclofenac Paracetamol Chlorzoxazone

1 3481243 9580728 141479182 3480070 9596079 141922753 3474685 9605600 142006134 3456683 9587599 141480735 3462315 9597724 14154756

Average 3470999 9593546 14168727SD 10967.78 9602.586 25622.336

% RSD 0.3 0.1 0.2Method Precision:Six test preparations were prepared as per the proposed test method for individual test preparation. All individual testpreparations were injected into the HPLC system as per the test method. The %assay results were calculated for eachindividual test sample, along with average assay and % RSD for the six preparations. The results are tabulated in Table 3.

Table 3: Method Precision Studies

Sr. No % AssayAceclofenac Paracetamol Chlorzoxazone

1 98.4 95.8 103.42 98.1 95.9 103.43 98.7 95.8 103.54 98.6 96.1 103.95 98.2 95.9 103.56 98.8 95.6 103.2

Average 98.5 95.9 103.5SD 0.2805 0.1643 0.2317

% RSD 0.3 0.2 0.2Ruggedness (Intermediate precision):Six test preparations were prepared as per the proposed test method for individual test preparation and injected into thedifferent HPLC system by the different analyst using different make of HPLC Column at different day.The %assay results were calculated for each of the sample for each of the variability. Average assay values and % RSD forthe six preparations were calculated. The results are tabulated in Table 4.



Table 4: Ruggedness


1 98.8 95.8 103.82 98.4 95.7 103.73 98.6 95.8 103.84 98.8 96.1 104.05 98.9 96.0 103.96 98.9 96.0 103.9

Average 98.7 95.9 103.9SD 0.1966 0.1549 0.1049

% RSD 0.2 0.2 0.1Table 5: Ruggedness data evaluation


1 98.4 98.8 95.8 95.8 103.4 103.82 98.1 98.4 95.9 95.7 103.4 103.73 98.7 98.6 95.8 95.8 103.5 103.84 98.6 98.8 96.1 96.1 103.9 104.05 98.2 98.9 95.9 96.0 103.5 103.96 98.8 98.9 95.6 96.0 103.2 103.9

OverallAverage 98.6 95.9 103.7

Overall SD 0.2697 0.1545 0.2570Overall% RSD 0.3 0.2 0.2

Linearity and Range:

Linearity of Detector Response:The linearity studies of detector response for analytes were evaluated in the concentration range from about 50% of lowerstrength to 150% of the higher strength of the targeted concentration. The diluted standard solutions were prepared fromstock solution in the above range and analysed using proposed analytical method by injecting each level in to the system.The Linearity graph of average area response verses concentration was plotted and the correlation coefficient wascalculated. The results are tabulated in Table 6.

Table6: Linearity of Detector Response:

Sr. NoAceclofenac Paracetamol Chlorzoxazone

Concentration(g/mL)

Arearesponse

Concentration(g/mL)

Arearesponse

Concentration(g/mL)

Arearesponse

1 29.9976 1051778 150.4635 2644943 120.2281 36036322 59.9951 2105682 200.6180 3519271 200.3802 60477723 75.9938 2451169 401.2361 7010633 320.6083 96117414 99.9919 3503313 501.5451 8745948 500.9505 149467985 109.9911 3849570 551.6996 9595498 601.1406 178849436 119.9903 4187013 626.9314 10901381 721.3687 213237307 159.9870 5600698 752.3177 13044334 761.4448 22494050

CorrelationCoefficient 0.999 1.000 1.000

Slope 35288.4907 17291.5590 29427.4152Intercept 56878.5049 55950.5535 139465.4305



Linearity of Test Method (Inferred from Accuracy):The results obtained in accuracy study were further interpreted for the evaluation of linearity of the test method. Theresults of average of mg added at each spiked level was plotted against average mg recovered at each accuracy level and thecorrelation coefficient for set of data was evaluated. The results are tabulated in Table 7.

Table 7: Linearity of Test method (Inferred from accuracy)For 100/500/500 mg Tablets:

Recovery level

Aceclofenac Paracetomol Chlorzoxazone

AmountAdded (mg)

AmountRecovered

(mg)

AmountAdded(mg)

AmountRecovered

(mg)

AmountAdded(mg)

AmountRecovered

(mg)80% 79.4 81.0 397.2 401.0 397.1 403.0

100% 99.6 101.0 495.8 498.0 495.6 502.0120% 119.6 121.0 594.8 590.0 595.0 594.0

CorrelationCoefficient 1.000 0.9999 0.9997

Slope 0.995 0.9565 0.9651Intercept 1.9627 21.9933 21.0867

Precision at Lower and Higher extreme concentrations:Precision at lower and higher extreme range concentration for Aceclofenac (29.9976 µg/ml and 159.9870 µg/ml),Paracetamol (150.4635 µg/ml and 752.3177 µg/ml), and Chlorzoxazone (120.2281 µg/ml and 761.4448 µg/ml) ofLinearity levels were determined as per proposed method by injecting six replicate injections of standards for both thelevels. % RSD for peak responses for both the level was evaluated. The results are tabulated in Table 8.

Table 8: Precision at Lower and Higher extreme Levels of linearityInjection Number

Area Responses Aceclofenac Area Responses Paracetamol Area ResponsesChlorzoxazone

Lower level(25%)

Higher level(150%)

Lower level(25%)

Higher level(150%)

Lower level(25%)

Higher level(150%)

1 1051198 5593151 2654481 13053921 3607531 224874582 1053062 5599304 2655088 13029744 3605991 224757483 1052477 5593984 2646499 13073632 3602871 225549934 1051003 5598529 2637079 13028568 3600717 224720925 1052376 5602959 2635339 13056944 3598395 225061956 1050552 5616261 2641174 13023193 3606288 22467814

Average 1051778 5600698 2644943 13044334 3603632 22494050SD 993.4749 8438.0317 8539.324 20088.1667 3586.7515 32895.1346

% RSD 0.1 0.2 0.3 0.2 0.1 0.1Accuracy:An accuracy study was conducted by spiking the known amount of analytes in the equivalent weight of placebo. Accuracystudy was conducted in triplicate at three different levels, (80%, 100% and 120% of target concentration). The sampleswere analyzed as per the proposed test procedure and the % recovery for each spike level was calculated. The precision ateach spike level was also established.The results are tabulated in Table 9, 10 and 11.



Table 9: Accuracy of AceclofenacRecovery

Level

AceclofenacAmountAdded (mg)

AmountRecovered (mg) % Recovery Average

Recovery (%) % RSD

80%79.4 80.5 101.4 101.5 0.479.4 80.9 101.979.5 80.4 101.1

100%99.6 101.7 102.1 101.7 0.399.6 101.1 101.599.6 101.2 101.6

120%119.6 119.8 100.2 101.3 1.0119.6 121.3 101.4119.6 122.4 102.3

Overall 101.5 0.6Table 10: Accuracy Paracetamol

RecoveryLevel

ParacetamolAmountAdded (mg)


Recovery (%) % RSD

80%397.1 399.9 100.7 101.0 0.4397.1 402.6 101.4397.3 400.3 100.8

100%495.8 500.1 100.9 100.3 0.5495.8 495.9 100.0495.9 496.5 100.1

120%594.8 582.6 97.9 99.2 1.3594.8 590.9 99.3594.8 597.3 100.4

Overall 100.2 1.0Table 11: Accuracy of Chlorzoxazone

RecoveryLevel

ChlorzoxazoneAmountAdded (mg)


Recovery (%) % RSD

80%397.1 403.6 101.6 101.6 0.5397.1 405.6 102.1397.2 401.1 101.0

100%495.6 502.6 101.4 101.4 0.1495.7 502.0 101.3495.6 502.4 101.4

120%595.0 588.2 98.9 99.8 1.2595.0 591.4 99.4595.0 601.3 101.1

Overall 100.9 1.0Robustness:

Effect of variation in Flow rate of mobile phase (±10%):Mobile phase Flow rate as per proposed analytical method is 1.0 ml/min. Change in flow rate by –10% = 0.9 ml/min. Change inflow rate by +10% = 1.1 ml/min. The effect due to change in flow rate on the system suitability parameters are compared.Results are tabulated in Table 12.



Table 12: System suitability of change in Flow RateSr.No. System Suitability parameter Observations LimitsAs Such - 10% + 10%

1

The % RSD ofpeak arearesponse for fivereplicateinjectionsAceclofenac 0.1 0.1 0.2 NMT 2.0Paracetamol 0.1 0.1 0.2Chlorzoxazone 0.1 0.2 0.3

2 Theoretical plates Aceclofenac 6209 6301 6158 NLT 1000Paracetamol 2559 2697 2413Chlorzoxazone 10476 10660 99883 Tailing factor Aceclofenac 2.0 2.0 2.0 NMT 2.0Paracetamol 1.4 1.4 1.3Chlorzoxazone 1.1 1.1 1.1

Effect of variation in Column oven temperature (±5°C):The Column Oven temperature as per proposed analytical method is 30°C. Change in Column oven Temperature by –5°C =25°C. Change in Column oven Temperature by +5°C = 35°C. The effect due to change in Column oven temperature on thesystem suitability parameters are compared. Results are tabulated in Table 13.

Table 13: System suitability of change in Column Oven temperatureSr.No. System Suitability parameter

ObservationsLimits

As Such -5°C + 5°C

1The % RSD of peakarea response for fivereplicate injections Aceclofenac 0.1 0.1 0.5 NMT 2.0Paracetamol 0.1 0.0 0.1Chlorzoxazone 0.1 0.0 0.1


Effect of variation in organic phase composition (± 10%):The Organic phase ratio of mobile phase as per proposed analytical method is 0.2% v/v Orthophosphoric acid: Acetonitrile is67:33 v/v. Change in Organic phase ratio of mobile phase by +10% and -10% of 0.2% v/v Orthophosphoric acid:Acetonitrile was performed. The effect due to change in organic phase composition on the system suitability parameters arecompared. Results are tabulated in Table 14.Table 14: System suitability of change in organic phase composition

Sr.No. System Suitability parameter Observations LimitsAs Such -5% + 5%

1The % RSD of peakarea response for fivereplicate injections Aceclofenac 0.1 0.2 0.1 NMT 2.0Paracetamol 0.2 0.1 0.2Chlorzoxazone 0.4 0.1 0.1




Effect of variation in Mobile phase pH (± 0.2 Units):The pH of mobile phase A as per proposed analytical method is 6.0. Change in pH of mobile phase A by – 0.2 units is 5.8Change in pH of mobile phase A by + 0.2 units is 6.2 The effect due to change in pH of mobile phase A on the systemsuitability parameters are compared. Results are tabulated in Table 15.Table 15: System suitability of change in mobile phase pH

Sr.No. System Suitability parameter

ObservationsLimits

As Such - 0.2 units + 0.2units

1

The % RSD ofpeak arearesponse for fivereplicateinjectionsAceclofenac 0.2 0.1 0.0 NMT 2.0Paracetamol 0.1 0.1 0.0Chlorzoxazone 0.2 0.1 0.1


Filter Interference studies:Standard solutions and sample solution were prepared. Some portion of above solutions was filtered through 0.45membrane filter. Both the samples further diluted as per proposed method and injected in to the HPLC system. Forcomparison the % area difference was calculated between unfiltered and filtered solutions. The data tabulated in Table 16,

17, 18 and 19.

Table 16: Filter paper detailsType Make Micron size Diameter Lot NoNylon MDI Syringe 0.45µ 25 mm SN0850

Table 17: Filter paper interference study Aceclofenac

Aceclofenac

Prep. No.Average Standard Area Average Test Area

STD-1 % Diff STD-2 %Diff Test-1 % Diff Test-2 %

DiffUnfiltered 3551905 NA 3524335 NA 3595532 NA 3591554 NA0.45µFilter 3548229 0.1 3563626 1.1 3588334 0.2 3581025 0.3

Table 18: Filter paper interference study ParacetamolParacetamol


STD-1 %Diff STD-2 % Diff Test-1 % Diff Test-2 %




Table 19: Filter paper interference study ChlorzoxazoneChlorzoxazone


STD-1 % Diff STD-2 %Diff Test-1 % Diff Test-2 %


Peak Purity results of Sample As such:Test preparation was prepared as per the test method and injected into HPLC system. The peak purity result wasevaluated by Photo Diode Array Detector.The results are tabulated in Table 20 and 21.

Table 20: Sample As such Higher Strength 100/500/500 mg tabletsPeak purity results

Parameter Aceclofenac Chlorzoxazone ParacetamolPurity-1 angle 0.071 0.271 2.092Purity-1 threshold 0.251 0.282 6.619Purity Flag No No No

Table 21: Sample As such Lower Strength 100/500/15 mg tabletsPeak purity results

Parameter Aceclofenac ParacetamolPurity-1 angle 0.057 2.171Purity-1 threshold 0.232 6.775Purity Flag No No

Forced Degradation studies:The stress degradation study was carried out on the sample preparations (100/500/500 mg and 100/500/15 mg strength)of Tablet, and the degradation was evaluated by calculating the % degradation of in comparison with unstressed samplepreparation. The degradation of 10-30% was tried by following stress conditions to prove the stability indicatingcharacteristics of the method. The stress conditions and results were compiled in Table 22 and 23.

Table 22: % degradation data for 100/500/500 mg tabletsStress Condition

Aceclofenac Paracetamol Chlorzoxazone%

degradation%

degradation%

degradationAcid degradation0.1N HCl, 1hr at 60°C 16.2 0.1 3.2Alkali degradation1N NaOH, 1hr at 80°C 95.1 9.9 7.0Peroxide degradation6% H2O2, 2hrs at 80°C 17.5 0.5 1.9Thermal degradation2 hrs at 80°C 11.3 1.4 1.4Photolytic degradation1.2 m.lux hrs 12.0 1.4 1.6



Table 23: % degradation data for 100/500/15 mg tabletsStress Condition

Aceclofenac Paracetamol%

degradation%

degradationAcid degradation 0.1N HCl, 1hr at 60°C 10.7 3.6Alkali degradation 1N NaOH, 1hr at 80°C 98.6 19.2Peroxide degradation 6% H2O2, 2hrs at 80°C 17.4 2.5Thermal degradation 2 hrs at 80°C 8.2 3.0Photolytic degradation 1.2 m.lux hrs 8.2 3.0Solution Stability Studies:

Mobile phase stability at room temperature:The mobile phase was prepared as per the proposed test method and kept at room temperature for a period of two days inwell closed condition. The system suitability solutions were prepared and injected into the HPLC system at initially andperiodically after 1day. The system suitability parameters were evaluated. The mobile phase was also observed for Physicalchanges like haziness or precipitation. System suitability results are tabulated in Table 24.

Table 24: Mobile phase stability at room temperatureSr. No. System Suitability parameter Observations LimitsInitial Day-1

1The % RSD of peak arearesponse for five replicateinjections Aceclofenac 0.2 0.1 NMT 2.0Paracetamol 0.2 0.2Chlorzoxazone 0.1 0.1

2 Theoretical plates Aceclofenac 6737 6704 NLT 1000Paracetamol 3013 3227Chlorzoxazone 10870 107253 Tailing factor Aceclofenac 1.9 1.8 NMT 2.0Paracetamol 1.3 1.2Chlorzoxazone 1.0 1.0

Analytical Solution Stability (Standard and Test preparation) at room temperature:Standard and test preparations were prepared as per the proposed test method and the stock solutions were kept at roomtemperature. The solutions were diluted freshly at each time and injected into the HPLC system at initially and at differenttime intervals. The % difference in area was calculated against the fresh standard solution injected at each time interval.The % difference in area response was evaluated against the initial assay value. The results are tabulated in Table 25, 26

and 27.

Table 25: Stability of Aceclofenac Standard and test solution at room temperatureAceclofenac Standard Aceclofenac Test

Time (Hours) Area Response % Difference Area Response % DifferenceInitial 3549534 NA 3598615 NA

4 Hrs 35 min 3539237 0.3 3578216 0.68 Hrs 3540881 0.2 3609559 0.3

12 Hrs 15 min 3559542 0.3 3597509 0.016 Hrs 20 min 3546836 0.1 3575222 0.720 Hrs 25 min 3540089 0.3 3575272 0.6

23 Hrs 3557104 0.2 3571046 0.824 Hrs 30 min 3528644 0.6 3562606 1.0

29 Hrs 3532159 0.5 3581784 0.533 Hrs 3581358 0.9 3581157 0.5

40 Hrs 50 min 3630421 2.3 3581119 0.5



Table 26: Stability of Paracetamol Standard and test solution at room temperatureParacetamol Standard Paracetamol Test

Time (Hours) Area Response % Difference Area Response % DifferenceInitial 9533772 NA 9674499 NA

4 Hrs 35 min 9485553 0.5 9681641 0.18 Hrs 9529119 0.0 9720005 0.5

12 Hrs 15 min 9562369 0.3 9713140 0.416 Hrs 20 min 9558274 0.3 9703185 0.320 Hrs 25 min 9551274 0.2 9705705 0.3

23 Hrs 9554327 0.2 9673281 0.024 Hrs 30 min 9551111 0.2 9659821 0.2

29 Hrs 9541020 0.1 9661378 0.133 Hrs 9619965 0.9 9674547 0.0

40 Hrs 50 min 9760962 2.4 9661793 0.1Table 27: Stability of Chlorzoxazone Standard and test solution at room temperature

Chlorzoxazone Standard Chlorzoxazone TestTime (Hours) Area Response % Difference Area Response % Difference

Initial 14101688 NA 14905697 NA4 Hrs 35 min 14074515 0.2 14896823 0.18 Hrs 14105493 0.0 15018775 0.812 Hrs 15 min 14182706 0.6 14980781 0.516 Hrs 20 min 14175597 0.5 14922091 0.120 Hrs 25 min 14185354 0.6 14925356 0.123 Hrs 14165758 0.5 14885819 0.124 Hrs 30 min 14150058 0.3 14871542 0.229 Hrs 14064098 0.3 14908388 0.033 Hrs 14290784 1.3 14911371 0.040 Hrs 50 min 14499323 2.8 14930540 0.2

BLANK CHROMATOGRAM



STANDARD CHROMATOGRAM

TEST CHROMATOGRAM

LINEARITY OF DETECTOR RESPONSE OF ACECLOFENAC



LINEARITY OF DETECTOR RESPONSE OF PARACETAMOL

LINEARITY OF DETECTOR RESPONSE OF CHLORZOXAZONE

LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF ACECLOFENAC



LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF PARACETAMOL

LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF CHLORZOXAZONE

AS SUCH SAMPLE CHROMATOGRAM HIGHER STRENGTH



AS SUCH SAMPLE PURITY PLOT HIGHER STRENGTH

AS SUCH SAMPLE CHROMATOGRAM LOWER STRENGTH



AS SUCH SAMPLE PURITY PLOT LOWER STRENGTH

ACID DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH



ALKALI DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

PEROXIDE DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

THERMAL DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH



PHOTOLYTIC DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

CONCLUSIONThe complete study results reveals that the developed and validated RP-HPLC method is accurate, precise, robust andstability indicating. This method has wide applicability and useful for regular quality control analysis of formulation samples.REFERENCES1. Granberg RA, Rasmuson AC, Solubility of paracetamol in pure solvents, Journal of Chemical & Engineering Data, 1999, 44(6), 1391–1395.2. Acetaminophen Drugs.com3. Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S, Paracetamol: new vistas of an old drug, CNS drug reviews,2006, 12 (3–4), 250–275.4. Altinoz MA, Korkmaz R, NF-kappaB, macrophage migration inhibitory factor and cyclooxygenase-inhibitions as likelymechanisms behind the acetaminophen- and NSAID-prevention of the ovarian cancer, Neoplasma, 2004, 51 (4), 239–247.5. Moller, P, Sindet-Pedersen S, Petersen C, Juhl G, Dillenschneider A, Skoglund L, Onset of acetaminophen analgesia:comparison of oral and intravenous routes after third molar surgery, British journal of anaesthesia, 2005, 94 (5): 642–648.6. Viswanathan, Feskanich, Schernhammer ES, Aspirin, NSAID and Acetaminophen use and the Risk of Endometrial Cancer,Cancer Research, 2008, 68 (7), 2507.7. Acetaminophen, chemicalland21.com, 2011.8. Byrant, Bronwen, Knights, Katleen, Salerno, Evelyn, Pharmacology for health professionals, Elsevier, 2007, 270.9. International Conference on Harmonization, Q2A: Text on Validation of Analytical Procedures, Federal Register, 1995, 60(40), 11260–11262.10. FDA, Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation, Availability,Federal Register (Notices), 2000, 65(169), 52776–52777.11. International Conference on Harmonization, Q2B: Validation of Analytical Procedures: Methodology andAvailability, Federal Register, 1997, 62 (96), 27463–27467.

12. USP 25–NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal Convention, Rockville,Maryland, USA, 2002), 2256.

Single Rp-hplc Method for the Quantification of Aceclofenac, Paracetamol and Chlorozoxazone in...

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