INTRODUCTION:

Aceclofenac (ace) and paracetamol (para) are non steroidal anti-inflammatory class of drugs. Paracetamol is official in IP¹, BP² and USP³. Chlorzoxazone (Chz) is a skeletal muscle relaxant which is official in USP. Aceclofenac is official in European Pharmacopoeia.⁴Aceclofenac, paracetamol and chlorzoxazone are available in combined tablet dosage form as analgesic agent. Aceclofenac relieves pain by stimulating cartilage synthesis, paracetamol inhibits prostaglandin synthesis and chlorzoxazone inhibits histamine release. Literature survey reveals that few UV^5,6, HPLC^7,8, HPTLC⁹ and colorimetric¹⁰ methods have been reported for the estimation of aceclofenac, chlorzoxazone and paracetamol as single component formulations and combination with other drugs in bulk samples, formulations and biological fluids. However not a single UV, HPLC, HPTLC method is reported for the simultaneous analysis of the three drugs in bulk and combined dosage form. Fixed dose combination of aceclofenac (100mg), chlorzoxazone (250mg) and paracetamol (500mg) is available in tablet form in the market.

The present study describes the determination of aceclofenac, chlorzoxazone and paracetamol in bulk drug sample and pharmaceutical dosage form using RP-C8 column with UV detection at 230.0nm. The method was validated according to procedures and acceptances criteria based on FDA guidelines and recommendations of ICH^{11, 12}.

MATERIAL AND METHODS:

A gradient HPLC (Merck Hitachi) with L-7100 double reciprocating pump, L-7400 UV detector and RP-Kromacil C8 column (250mm×4.6mm, i.d: 5µm) was used with Winchrome software. Aceclofenac, chlorzoxazone and paracetamol were obtained as gift samples from Que Pharmaceuticals Ltd., Gujarat, India. Acetonitrile used was of HPLC grade (Qualigens). All other reagents used in the study were of AR grade (Qualigens).

PROCEDURE:

Preparation of stock solution of Aceclofenac, chlorzoxazone and paracetamol:

About 10 mg of aceclofenac, chlorzoxazone and paracetamol each was accurately weighed and transferred to 100 ml volumetric flasks respectively. It was dissolved in acetonitrile and double distilled water in the ratio of 60:40 and the solution was made up to the volume with same to obtain 100µg/ml of aceclofenac, chlorzoxazone and paracetamol respectively. From this standard stock solution, the mixed standard solution was prepared to contain 4µg/ml of aceclofenac, 10 µg/ml of chlorzoxazone and 20 µg/ml of paracetamol.

Chromatographic Conditions:

The mobile phase consisting of Acetonitrile : Double distilled water (60:40) was filtered before use through 0.45 µ membrane filter and was pumped by the dual plunger reciprocating pump (L-7100 Lachrom, Hitachi) at a flow rate of 1 ml/min in the ratio of 60:40. The separation was carried out on a C8 column (5µm, 250 (L) X 4.6 mm i.d., Kromasil) attached with a C8 guard column. The column temperature was maintained at 28⁰C. The sample was injected through a Rheodyne injector and was analyzed by variable wavelength detector set at 230.0 nm. The data was acquired, stored and analyzed using winchrom software.

Table 1: Analysis of TABLET formulation

Component	Amount Present (mg)	Amount Found* (%)	Standard Deviation*	% RSD*	Standard Error*
ACE	100	98.43	0.4536	0.4608	0.1852
CHZ	250	99.9	0.5155	0.5160	0.2104
PARA	500	100.09	0.1724	0.1722	0.07040

*Denotes average of six determinations.

ACE, CHZ and PARA for Aceclofenac, Chlorzoxazone and Paracetamol respectively. ZACY- MR (Comed Pharmaceuticals).

Procedure:

The standard stock solution of each drug was suitably diluted with the mobile phase to obtained standard solutions of different concentrations. Each standard solution was injected six times into the column at a flow rate of 1 ml/min. The good linearity was obtained in the concentration range 1-60 µg/ml for ACE, 1-50 µg/ml for CHZ and PARA.

Assay of tablet formulation:

Twenty tablets were weighed accurately and the average weight was determined and triturated to a fine powder. A quantity equivalent to 100mg of ACE, 250mg CHZ and 500 mg of PARA was weighed and transferred to a 100 ml volumetric flask. The contents were sonicated for 20 min with mobile phase to dissolve the active ingredients and the volume was made up to 100 ml with same and filtered through a 0.2 µ membrane filter. Suitable aliquots of the solution were further diluted with mobile phase to obtain sample solutions within the concentration range for the three drugs. Each sample solution was injected into sample injector with 20µl loop of HPLC six times under the chromatographic conditions as described above. The area under the curve of each peak was measured at 230.0 nm. The amounts of each drug present in the sample solutions were determined using the prepared calibration curves of standard ACE, CHZ and PARA respectively.

The developed HPLC method was used to quantify ACE, CHZ and PARA in the formulation. The % drug content was found to be 98.43, 99.9 and 100.09 of the labeled amount for ACE, CHZ and PARA respectively. The result of formulation analysis and its statistical analysis are given in Table No.1 indicates high degree of precision of the proposed method. The proposed HPLC method was also validated as per ICH guidelines.

Precision:

Intra day and Inter day precision was determined by repeating assay three times in same day for intra day and on different day for inter day precision (Table No.3).

Accuracy:

To check the accuracy of the proposed method, recovery studies were carried out at 80, 100 and 120 % of the test concentration as per ICH guidelines. The results of the recovery studies and its statistical validation data given in Table No.2 indicate high accuracy of the proposed method.

Robustness:

To evaluate the robustness of the developed method, deliberate variations were made in the method parameters such as the flow rate, ratio of mobile phase and the column temperature.

Specificity:

The specificity of the HPLC method was ascertained by analyzing standard drug and sample solutions. The retention time of ACE, CHZ and PARA was confirmed by comparing the retention time with that of the standard.

LOD and LOQ:

The LOD and LOQ were separately determined based on the standard deviation of response of the calibration curve. The residual standard deviation of the regression lines and slope of the calibration curves were used to calculate the LOD and LOQ (Table No. 3).

Fig:1 Typical Chromatogram of Aceclofenac,Chlorzoxazone and Paracetamol (Aceclofenac-2.00 min. , Chlorzoxazone-2.95 min. and paracetamol – 4.38min.)

RESULTS AND DISCUSSION:

The multicomponent formulations have gained a lot of importance as there is greater patient acceptability, increased potency and decreased side effect. Aceclofenac, chlorzoxazone and paracetamol is a recent combination in the market used for pain relief. The goal of this study was to develop a rapid and sensitive HPLC method for the analysis of ACE, CHZ and PARA in bulk drug samples and its formulation using the most commonly employed RP-C8 column with UV detection. The mobile phase consisted of acetonitrile and double distilled water in the ratio of 60:40.

TABLE NO. 2 Recovery studies and ITS statistical validation data

Level of % Recovery	Component	Amount present (mg)	Amount of Standard added (mg)	% Recovery*	Standard deviation*	% RSD*	Standard error*
80	ACE	100	80	99.34	0.3523	0.3546	0.2034
	CHZ	250	200	100.36	0.1155	0.11508	0.0667
	PARA	500	400	99.47	0.6200	0.6233	0.3580
100	ACE	100	100	98.91	0.1443	0.1458	0.0835
	CHZ	250	250	98.8	0.0500	0.0506	0.02887
	PARA	500	500	99.95	0.1803	0.1803	0.1041
120	ACE	100	120	99.70	0.8776	0.8880	0.5067
	CHZ	250	300	98.02	0.02309	0.02355	0.01333
	PARA	500	600	100.6	0.2082	0.2069	0.1202

* Denotes average of three determinations at each level of recovery.

TABLE: 3 VALIDATION AND SYSTEM SUTIABILITY STUDIES

Parameter	Aceclofenac	Chlorzox azone	Paracet amol
Linearity range (µg/ml)	1-60 µg/ml	1-50 µg/ml	1-50 µg/ml
Intercept	224702.66	202329.33	15332.66
Slope	585453.66	510917	549778.33
Correlation coefficient (%) R.S.D	0.9995 0.5882	0.9995 0.5596	0.9996 0.5450
Limit of detection (µg/ml)	0.0166	0.0194	0.0011
Limit of quantitation (µg/ml)	0.050	0.0599	0.0034
Retention time (min.)	2.00	2.95	4.38
Robustness	Robust	Robust	Robust
Precision (% R.S.D) Inter-day (n=3) Intra day (n=3)	0.4473 0.5296	0.5082 0.6323	0.5411 0.2000
Tailing factor	1.12	1.10	1.10
Theoretical Plates	984.64	1205.2	2657.0
Mean % recovery	99.31	99.06	100.01
Resolution factor	Between ACE and PARA is 3.8, Between PARA and CHZ is 5.72

The run time was set at 10 min and the retention times for ACE, CHZ and PARA were 2.00, 2.97 and 4.38 min. respectively (Fig No. 1). The peak areas of both the drugs were reproducible as indicated by RSD values less than 2%. When the calibration curve for concentration of aceclofenac, chlorzoxazone and paracetamol and its respective peak areas were plotted, a good linear relationship was observed between the concentration and their respective peak areas in the range of 1-60 µg/ml for ACE and 1-50 µg/ml for both CHZ and PARA respectively. The results of the formulation analysis, recovery studies and its statistical validation data given in Table No.1 and 2 indicate high degree of precision and accuracy of the proposed method. The results of the validation and system suitability parameter are given in Table No.3 Hence it can be concluded that the developed RP-HPLC method can be employed successfully for the estimation of Aceclofenac, cholzoxazone and paracetamol in both bulk and multicomponent formulation.

ACKNOWLEDGEMENT:

The authors thank Que Pharmaceuticals Pvt. Ltd. Wadhwan Gujarat, for supplying gift samples of Aceclofenac, chlorzoxazone and paracetamol to carry out this work.

REFERENCES:

1. Indian Pharmacopoeia, New Delhi, 1996(I), pp. 554.

2. British Pharmacopoeia, H. M .Stationary Office, London, 2004, (II), pp.48.

3. The United States Pharmacopoeia 27, National Formulary 22.Convention Inc.1260, Twinbrook Parkway, Rockville, MD.20852, 2004, pp.441

4. S.C.Sweetman., Martindale: The Complete Drug Reference; 34^th Edn. The Pharmaceutical Press, London, 2005, pp. 21,73,1313.

5. Srivasan K.K., Shirwarkar A. et.al, Simultaneous estimation of Aceclofenac and paracetamol in solid dosage form by UV spectrophotometry, Indian drugs, Feb 2006, 43(2), 141-145.

6. Ali M.S., Rafiuddin S., Simultaneous determination of paracetamol, chlorzoxazoneand related impurities 4-aminophenol, 4-chlorophenol, 4-chloroacetanilide and p-chlorophenol in pharmaceutical preparation by HPLC, J.AOAC Int, Jan-Feb 2007, 90(1), 82-93.

7. M.K. Chouksey et al. Simultaneous estimation of paracetamol, chlorzoxazone and diclofenac Na in dosage forms by HPLC method, Indian drugs2006, 43(3), pp.216-220.

8. B.Hinz et al. Simultaneous determination of Aceclofenac and three of its metabolites in human plasma by HPLC, Biomed Chromatography, 17(4), 2003, pp. 268-275.

9. El-Sharty Y.S., Refaat M., Drug Dev. Ind. Pharm, May 2002, 28(5), 571-582.

10. Zawilla N.H., Mohammad M.A. et.al. J.Pharma Biomed Analysis, Jan 2002, 27(1-2), 243-251.

11. ICH, Q2A, Text on Validation of Analytical Procedures, International Conference on Harmonization, Geneva, October, 1994, pp.1-5.

12. ICH, Q2B, Validation of Analytical Procedures: Methodology, International Conference on Harmonization, Geneva, November, 1996, pp.1-8.

Received on 24.10.2008 Modified on 14.11.2008

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 161-163

Tailing factor