Asian J. Research Chem.. 2(1): Jan..-Mar.

2009 ,

ISSN 0974-4169 RESEARCH ARTICLE

www.ajrconline.org

Reverse Phase HPLC Method for Determination of Aceclofenac and Paracetamol in Tablet Dosage Form
Dept. of Pharmaceutical Analysis, Seth Govind Raghunath Sable College of Pharmacy,Saswad, Pune- 412 301, India * Corresponding Author E-mail: godse_vijaya@rediffmail.com A simple, rapid and selective HPLC method has been developed for quantitation of aceclofenac and paracetamol from bulk drug and pharmaceutical formulations using a mobile phase consisting mixture of methanol and water (70:30 v/v) at the flow rate of 1mL/min. An ODS C-18 (Intersile 25 cm x 4.6 mm, 10 m.) column was used as stationary phase. The retention time of aceclofenac and paracetamol were 1.8 min. and 2.7 min. respectively. Linearity was observed in the concentration range of 2-50 g/mL for aceclofenac and 5-50 g/mL for paracetamol. Percent recoveries obtained for aceclofenac and paracetamol were 100.6 and 100.7 respectively. The proposed method is precise, accurate, selective and rapid for the simultaneous determination of aceclofenac and paracetamol.

Godse VP.* Deodhar MN, Bhosale AV, Sonawane RA, Sakpal PS, Borkar DD and Bafana YS

ABSTRACT

KEY WORDS INTRODUCTION:

Aceclofenac, Paracetamol, HPLC Method Apparatus and Chromatographic Conditions: Chromatographic separation was performed on a Jasco HPLC system consisting of Jasco PU-2080 pump, Jasco UV 2010 photo diode array detector, Rheodyne injection syringe with 20 l loop volume and windows based chrompass software. An ODS C18 RP-Column (Intersile 4.6 mm x 25 cm, 10 m) was used for separation. The elution was carried out isocratically at flow rate of 1 mL/min using methanol: water (70:30 v/v) mobile phase. Preparation of Standard Stock Solution: The standard stock solution 1mg/ml of aceclofenac and paracetamol were prepared separately by dissolving 50 mg of each drug in 50 ml mixture of methanol and water (70:30 v/v). From the standard stock solution, mixed standard solution was prepared to contain 100 g/mL of aceclofenac and 100 g/mL of paracetamol.
Table 1. Linearity Aceclofenac Concentration Peak area (g/mL) (v) 2 1364.8 5 4839.7 10 9430.2 20 19261.6 30 28712.0 40 30707.3 50 46484.5 Paracetamol Concentration (g/mL) 5 10 20 25 30 40 50

Aceclofenac is chemically designated as 2-(2, 6dichlorophenyl) amino phenyl acetyl oxyacetic acid. It is used as anti-inflammatory and analgesic agent. Paracetamol is chemically N - (4-hydroxyphenyl) acetamide and is used as analgesic and anti-pyretic agent. Detailed survey of literature for paracetamol revealed several methods based on techniques viz. HPLC1-5, spectrophotometric6-10 for its determination in pharmaceutical dosage form and in human plasma. Similarly, survey of literature for aceclofenac revealed several methods based on HPLC11-15 spectrophotometric16 and HPTLC17 for its determination in human plasma and in pharmaceutical formulations. However, no method has been developed for estimation of these drugs in combined dosage form. This paper presents simple, rapid, reproducible and economical method for RPHPLC simultaneous estimation of aceclofenac and paracetamol in tablet dosage form.

EXPERIMENTAL:

Reagents and Chemicals: Methanol HPLC grade was procured from Sisco Research Laboratories Pvt. Limited, Mumbai. Water HPLC grade was obtained from Merck Specialties Private Limited, Mumbai. Reference standards of aceclofenac and paracetamol were procured from Emcure Pharmaceuticals Ltd, Pune.

Peak area (v) 2575.1 4409.3 10141.6 12579.0 15260.9 20089.1 24767.9

Received on 04.03.2009 Accepted on 23.03.2009

Asian J. Research Chem. 2(1): Jan.-March, 2009;Page 37-40

Modified on 11.03.2009 AJRC All right reserved

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Table 2. Analysis of formulation

Asian J. Research Chem.. 2(1): Jan..-Mar. 2009 ,

Drug Labeled Amount mg/tab Found* Aceclofenac 100 98.0 Paracetamol 500 499.2 * Average of six determinations

% Label Claim* 98.72 99.46

Standard deviation 0.8239 0.2324

% R.S.D. 0.8346 0.2337

Standard Error 0.4762 0.1343

Preparation of Sample Solution Twenty tablets (Afenak Plus, Ranbaxy Lab, Mumbai) each containing 100 mg of aceclofenac and 500 mg of paracetamol were weighed and powder equivalent to 100 mg of paracetamol and 20 mg of aceclofenac was weighed accurately (0.1002 gm ) and transferred to a conical flask and extracted thrice with 20 ml mixture of methanol and water (70:30). The combined extracts were transferred to a volumetric flask and the volume adjusted to 100 ml with mobile phase. From this solution, 10 ml was pipetted and transferred to 100 ml volumetric flask and made volume up to the mark with mobile phase to get the concentration 100 g/ml of paracetamol and 20 g/ml of aceclofenac. Further dilutions were made using mobile phase to get the final concentration of 10 g/ml of paracetamol and 2 g/ml aceclofenac Linearity Several aliquots of standard solutions of aceclofenac and paracetamol were taken in different 10ml volumetric flasks and diluted up to the mark with mobile phase such that the final concentration of aceclofenac and paracetamol is 2-50 g/mL and 5-50 g/mL respectively. Evaluation of two drugs were performed with PDA detector at 275 nm, peak area recorded for all the peaks and are given in the Table I. The slope and intercept value for calibration curve was y = 947.82x + 1.1033 (R2 = 0.9989) for aceclofenac and y = 502.37x + 84.858 (R2 = 0.09987) for paracetamol. The results show that an excellent correlation exists between peak area and concentration of drugs within the concentration range indicated above.
Table 3. System suitability parameters for tablet analysis Parameters Theoretical plates Resolution factor Asymmetric factor LOD (ng/ml) LOQ (ng/ml) Aceclofenac 3181.735 --1.42 5 10 Paracetamol 6924.0525 2.07 2.18 10 20

Estimation of Aceclofenac and Paracetamol in Dosage Forms: The HPLC procedure was optimized with a view to develop accurate and stable assay method. Both the pure drugs aceclofenac and paracetamol were run in different mobile phase compositions with different C18 columns [Kromacil 25cm x 4.6mm i.d. 5 micron), ODS C18(Intersile 25cm x4.6mm i.d.,10 micron)]. The flow rate was also varied from 0.5 mL to 1.3 mL/min. ODS C18 column with a mobile phase of mixture of methanol and water (70:30 v/v), delivered at a flow rate of 1.0 mL/min with a detection at 275 nm gave sharp and symmetrical peak with retention time 1.8 and 2.7 min for aceclofenac and paracetamol respectively. The typical chromatogram of the sample is shown in Fig. 1.The overlaid UV spectrum of aceclofenac and paracetamol is shown in Fig. 2. The assay procedures were repeated for six times and the percentage of individual drugs found in formulations, standard deviation, % R.S.D. and standard error were calculated and presented in Table II.

RESULTS AND DISCUSSION:

Fig.1.-Chromatogram of aceclofenac and paracetamol

Assay 20 l of standard and sample solutions were injected into an injector of liquid chromatograph, from the peak area of aceclofenac and paracetamol amount of drug in samples were computed. The values are given in Table II.
Fig.2. - Overlain UV spectrum of aceclofenac and paracetamol

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Table 4. Recovery Study for Spiked Concentration of Drugs to the Pre-analyzed Dosage Forms Level of % Mean Recovery % Recovery* ACF PCM 50 100.77 100.66 100 101.00 99.50 * Average of Three determinations. Standard deviation ACF PCM 0.692 0.034 0.750 0.663 Co-efficient of Variation ACF PCM 0.687 0.034 0.742 0.659 Standard Error ACF PCM 0.400 0.020 0.433 0.255

Asian J. Research Chem.. 2(1): Jan..-Mar. 2009 ,

Method Validation: Limit of Detection and Limit of Quantification The limit of Detection (LOD) and limit of Quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed RP-HPLC method. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOD for aceclofenac and paracetamol was found to be 10 ng/ml and 5 ng/ml, respectively. The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10). The LOQ was 30 ng/ml and 15 ng/ml for aceclofenac and paracetamol respectively Table III. Ruggedness and Robustness The ruggedness of the method was determined by carrying out the experiment on different instruments like Jasco HPLC and Agilent HPLC by different operators using different columns of similar type like Hypersil C18, Intersile C18. Robustness of the method was determined by making slight changes in the chromatographic conditions. It was observed that there were no marked changes in the chromatograms, which demonstrated that the RP-HPLC method developed, are rugged and robust. Solution Stability In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 5 h at room temperature. The result show that for both solutions, the retention time and peak area of aceclofenac and paracetamol remained almost unchanged (% R.S.D. less than 2.0) and no significant degradation within the indicated period, thus indicated that both solutions were stable for at least 5 h, which was sufficient to complete the whole analytical process. System Suitability Studies The column efficiency, resolution and peak asymmetry were calculated for the standard solutions Table III. The values obtained demonstrated the suitability of the system for the analysis of this drug combination, system suitability parameters may fall within 3% standard deviation range during routine performance of the method. Recovery Studies To study the accuracy and reproducibility of the proposed method recovery experiments were carried out. A fixed amount of pre-analyzed sample was taken and standard drug was added at 50% and 100% levels. Each level was repeated three times. The contents of aceclofenac and

paracetamol per tablet found by proposed method is shown in Table IV. The lower values of RSD of assay indicate the method is accurate. The mean recoveries of aceclofenac and paracetamol were in range of 100.77% and 100.66% that shows there is no interference from excipients. The proposed method gives good resolution between aceclofenac and paracetamol within short analysis time (< 10min). The method is very simple, rapid and no complicated sample preparation is needed. High percent of recovery shows the method is free from interference of excipients present in the formulations. The authors are grateful to Emcure Pharmaceuticals, Pune and S.P.B. Ink Ltd, Pune for providing gift samples of aceclofenac and paracetamol respectively.

CONCLUSION:

ACKNOWLEDGEMENT:

REFERENCES:
1. Ravisankar S, Vasudevan M and Najan MJ. Reverse phase HPLC method for the estimation of Paracetamol, Chlorzoxazonze, and Diclofenac sodium in formulations. Indian Drugs.1997;34 (11):663-665. Shenoy KRP, Krishnamurthy KS and Sumatheendra KS. Determination of Codeine phosphate, oxylamine succinate, Paracetamol and Caffeine in combined dosage formulation by reverse phase liquid chromatography. Indian Drugs. 2000;37(10): 486-488. Shinde VM and Raman R. Simultaneous determination of paracetamol and chlormezanone in tablets by RP-HPLC. Indian Drugs.1998; 35 (8):521-523. Yong Y F and Wang AJ. Determination of pseudo ephedrine and paracetamol in their tablets by RP-HPLC. Yaouw Fenxi Zazhi.1998; 16(4):216-218. Vasudevan M, Ravisankar S, Ravibabu T and Nanjan MJ. Simultaneous estimation of Paracetamol, Methocarbamol and Ibuprofen by reversed phase HPLC method. Indian Drugs.2000;37(8):386-389. Gangwal S and Trivedi P. simultaneous analysis of Indomethacin and Paracetamol in combined dosage form by spectropphotometry. Indian Drugs.1998; 35(5):291-295. Bhatia MS, Kashedikar SG and Chaturvedi SC. Comparative evaluation of Different Spectrophotometric methods for Simultaneous estimation of Paracetamol, Chlorzoxazone and Diclofenac sodium in combined dosage forms, Indian Drugs.1997;34(3):49-153. Aditya N, Arora RK and Tiwari M. Simultaneous spectrophotometeric estimation of Valdicoxib and Paracetamol in tablet formulation. Indian J. Pharma. Sci. 2006;68(3):370-373.

2.

3. 4. 5.

6. 7.

8.

39

9. 10.

11. 12. 13.

14.

15. 16.

17.

Jin JR and Lin WH. Modifying the multiwavelength k factor spectrophotometry, Fenxi Shiyanshi.1995; 14(2):22-25. Milch G and Szabo E. Drivative spectrophotometric assay of acetaminophen (paracetamol) and spectroflurimetric determination of its main impurity. J. Pharm Biomed. Anal. 1991; 10:1107-1113. Gao LQ., Liu WY and Xing JD. Determination of aceclofenac in plasma by RP-HPLC with solid phase back- flushing extraction method. Yaouw Fenxi Zazhi, 1998; 18(4):238-241. Jin Y, Chen H and Zeng FD: Determination of aceclofenac in human plasma by RP-HPLC.Sepu.2004; 22(4):2-254. Aloba OT, Adusumilli PS and Nigalaye AG. HPLC analysis of multicomponent product using a silica stationary phase and an aqueous organic phase. J. Pharm Biomed. Anal. 1991;9(4):335-340. Lee HS, Jeong CK, Choi SJ, Kim SB and Lee MH. Simultaneous determination of aceclofenac and diclofenac in human plasma by narrow bore HPLC using column switching. J.Pharm Biomed. Anal. 2000;23(5):775-781 Kousey E, Zawilla NH and Abdul AM. Determination of aceclofenac in bulk and pharmaceutical formulation by HPLC. J. Pharm Biomed. Anal. 2002; 9(4):243-251. Shanmugam S, Cendil KA, Vetrichelvan J, Manavalan R, Venkappayya D and Pandey V. Spectrophotometric method for estimation of Aceclofenac in tablets, Indian Drugs.2005;42:106-107. Sane RT, Menon SN, Mote M and Inamdar S. HPTLC determination of aceclofenac in bulk drug and in pharmaceutical preparation. J. Planer Chromatogr. 2004;17(3):241-245

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