INTRODUCTION:

Paracetamol is N-(4-hydroxyphenyl) acetamide; commonly known as acetaminophen^1,2. It is a widely-used as analgesic and antipyretic medication. Derived from coal tar, it is the active metabolite of phenacetin, but unlike phenacetin, paracetamol has not been shown to be carcinogenic.It is available over-the-counter. It is commonly used for the relief of fever, headaches, and other minor aches and pains^3,4. In combination with non-steroidal anti-inflammatory drugs (NSAIDs) or opioid analgesics, paracetamol is used also in the management of more severe pain.

Diclofenc sodium is 2-(2-(2,6-dichlorophenylamino) phenyl) acetic acid. is a non-steroidal anti-inflammatory drug (NSAID) taken to reduce inflammation and as an analgesic in conditions such as arthritis or acute injury^1,2,4. It can also be used to reduce menstrual pain, dysmenorrhea^3,4.

Chlorzoxazone is 5-chloro-3H-benzooxazol-2-one. Chlorzoxazone is a centrally acting muscle relaxant used to treat muscle spasm and the resulting pain or discomfort^1,4. These drugs are official in IP, BP, and USP. HPLC method has been reported for this combination^5-7.

No HPTLC method has been reported for this multidrug combination. In recent years HPTLC technique has been improved to incorporate certain features automated sample application, controlled development environment, computer controlled densitometry etc.

Validation of an analytical procedure is done using the guidelines suggested by the International Conference on Harmonization [ICH Topic Q2 (R1)].^8,9 The validation of an analytical method development is an essential part after the method has been developed to check whether it is suitable for different conditions and with varying concentrations of the sample.^11,12 The aim of the present work is to develop and validate HPTLC method for estimation of Paracetamol, Diclofenac sodium, Chlorzoxazone in tablet dosage forms.

Table No.1 Results of Assay, LOD and LOQ values for Paracetamol, Diclofenac sodium and Chlorzoxazone in pharmaceutical dosage forms

Brand Name	Sr. No.	Amount Label claimed (mg)
Brand Name	Sr. No.	Paracetamol (500mg)	Diclofenac Sodium (50mg)	Chlorzoxazone (500mg)
CIP-ZOX	1	500.12	50.31	499.93
	2	499.06	49.67	500.21
	3	499.87	49.53	499.43
	4	499.63	49.93	499.84
	5	499.60	49.31	500.03
Mean assay		499.65 ± 0.60	49.75 ± 0.72	499.88 ± 0.40
R.S.D (%)		1.67	1.54	1.87
LOD		100	100	500
LOQ		300	300	1500

Table No 2: Results from precision evaluation

Drug	Concentration (ng/spot)	Intra-day precision ( % RSD, n=3)	Interday precision (% RSD, n= 3 )
Paracetamol	2000	0.252	0.563
	3000	0.793	0.931
	4000	0.382	1.23
Diclofenac sodium	3000	0.573	0.592
	4000	0.984	0.783
	5000	0.703	0.739
Chlorzoxazone	2000	1.04	0.963
	3000	0.232	0.762
	4000	0.653	0.547

Table No 3: Results from recovery studies

Component	Label claim (mg/tablet)	Initial amount (ng)	Amount added (ng)	Amount recovered (ng)	% recovered	% RSD
Paracetamol	500	3000	1500	1496	99.73	0.659
		3000	0	2993	99.76	0.874
		3000	4500	4502	100.04	0.459
Diclofenac sodium	50	4000	2000	1994	99.70	1.65
		4000	0	3995	99.87	1.36
		4000	6000	5999	99.98	1.05
Chlorzoxazone	500	3000	1500	1487	99.13	0.597
		3000	0	2999	99.96	0.542
		3000	4500	4504	100.08	1.466

Figure No.1 A typical chromatogram of Paracetamol, Diclofenac Sodium and Chlorzoxazone measured at 254nm. Mobile phase- Chloroform: Methanol: Ammonia (20:5:0.2v/v/v).

Figure No.2 Typical Absorption Spectra of Paracetamol, Diclofenac Sodium and Chlorzoxazone

MATERIALS AND METHODS:

· Instrumantation:

Camag (Muttenz; Switzerland) Linomat V applicator, Camag twin trough TLC chamber, Camag TLC scanner 3, Camag Wincats software and Hamilton Syringe (100µl)

· Materials and reagents:

Analytically pure gift samples of Paracetamol, Diclofenac sodium, Chlorzoxazone were obtained from Hetero Labs Ltd. Mumbai; India was used as working standard. Chloroform, Methanol and Ammonia of HPLC grade (Merck Chem.) to prepare mobile phase were used without further purification.

· Standard stock solutions:

Standard stock solutions containing 0.5mg/mL of all of the Paracetamol, Diclofenac sodium and Chlorzoxazone were prepared by dissolving 5mg standard Paracetamol, Diclofenac sodium and Chlorzoxazone in 10ml methanol in 3 different flasks and are used as working standard solutions.

· Sample preparation:

Twenty CIP-ZOX tablets manufactured by CIPLA containing 500mg of Paracetamol, 50mg of Diclofenac sodium and 500mg of Chlorzoxazone were weighed and powdered. An amount of powder equivalent to 500mg of Paracetamol, 50mg of Diclofenac sodium and 500mg of Chlorzoxazone was transferred to 250ml calibrated volumetric flask containing 100ml methanol sonicated for 15min. Then the resulting solution was filtered using 0.45µm filter. Take 1ml of solution from filtrate and dilute it for 10ml of methanol. Also take 4ml of solution from filtrate and dilute it for 10ml of methanol. A sample solution (6µL containing 3000ng of Paracetamol and Chlorzoxazone; 1200ng of Diclofenac sodium) was spotted for assay of Paracetamol, Diclofenac sodium, Chlorzoxazone.

· Chromatography:

Chromatography was performed on 20cm X 10cm aluminium backed silica gel 60 F₂₅₄HPTLC plates (Merck, Dermatadt, Germany). The plates were prewashed with methanol and dried in an oven at 50⁰C for 5min. Samples were applied as 6mm bands spraying at a rate of 15µL-1s by means of a Camag (Muttenz; Switzerland) Linomat V sample applicator equipped with a 100-µL syringe (Hamilton). The distance between the bands was 10.0mm. Ascending development of the plate, migration distance 80mm was performed at 25±2⁰C with Chloroform: Methanol: Ammonia (20:5:0.2v/v/v) as mobile phase in a Camag twin trough chamber previously saturated for 20min with paper. The average development time was 20min. Densitometric scanning was then performed with a Camag TLC scanner 3 equipped with Wincats Software Version 1.4.4 at λ=254nm using Deuterium light source, the slit dimensions were 5.00 X 0.45mm.

RESULT AND DISCUSSION:

Validation of HPTLC method: ^8-12

· Linearity:

Amounts of standard solutions equivalent to 1000-5000 ng/spot Paracetamol, Diclofenac sodium, Chlorzoxazone was spotted on prewashed HPTLC plates. The plate was developed, dried and scanned as described above. The calibration plot was constructed by plotting peak area against the corresponding concentrations of all the drugs (ng/spot) individually. The linearity of response for Paracetamol, Diclofenac sodium, Chlorzoxazone was assessed in the concentration range 1000-5000 ng/spot, in terms of intercept and correlation coefficient values. The calibration spots showed the correlation coefficients (r=0.9956, 0.9988, 0.9988), intercept were (2309, 2782, 1140) respectively over the concentration range studied with six replicate readings of each concentration.

· Sensitivity:

The sensitivity of measurement of Paracetamol, Diclofenac sodium and Chlorzoxazone by the use proposed method was estimated in terms of Limit of Quantitation (LOQ) and the lowest concentration detected under the chromatographic conditions as the Limit of Detection (LOD). The LOD and LOQ were calculated by the use of the equation LOD = 3 X N/B and LOQ = 10 X N/B where N is the standard deviation of the peak areas of the drugs taken as a measure of noise and B is the slope of the corresponding calibration curve. The results of sensitivity are as shown in Table No.1.

· Assay and Precision evaluation:

The amount of Paracetamol, Diclofenac sodium and Chlorzoxazone was applied in number of replicates of both pharmaceutical preparations (n = 6) and the assay results are reported in Table No.1. Precision studies were performed using standard solution of Paracetamol, Diclofenac sodium and Chlorzoxazone the concentration of drugs covering the entire calibration range. The precision of the method in terms of intra-day variation (% CV) was determined by analysing Paracetamol, Diclofenac sodium and Chlorzoxazone standard drug solution within the calibration range, three times on the same day. Inter-day precision (% CV) was assessed by analysing Paracetamol, Diclofenac sodium and Chlorzoxazone standard drug solution within the calibration range on three different days over a period of one week. The results of precision studies are as shown in Table No.2.

· Accuracy:

The accuracy of the method was determined by the use of standard additions at three different levels i.e. multiple level recovery studies. Sample stock solution of tablet formulation of 3000ng/spot of Paracetamol, Chlorzoxazone and 4000ng/spot of Diclofenac sodium was prepared from tablet solution and spiked with amounts equivalent to 50, 100. 150 % of amounts of drugs in the original solutions. When these solutions were analyzed the recoveries were found to be within the acceptable limits of 98-102%. The reports of recoveries are as shown in the Table No.3.

· Specificity:

The mobile phase designed for the method resolved the drugs very efficiently as shown in Figure No.1 The R_f values of Paracetamol, Diclofenac Sodium and Chlorzoxazone were found to be 0.53, 0.27 and 0.68 respectively. The typical absorption spectrum for Paracetamol, Diclofenac Sodium and Chlorzoxazone is shown in Figure No.2. The peak purity was tested by correlating the spectra’s of Paracetamol, Diclofenac Sodium and Chlorzoxazone at the peak start (S), peak apex (A) and peak end (E) positions. Correlation between these spectra’s indicated purity of Paracetamol, Diclofenac Sodium and Chlorzoxazone peak correlation r (S, A) = 0.9996, 0.9994, 0.9999, and r (A, E) = 0.9997, 0.9992, 0.9998. It can be thus concluded that no impurities or degradation products were found with the peaks of standard solution.

· System suitability:

According to USP system suitability tests are an integral part of a chromatographic analysis and should be used to verify that the resolution and reproducibility of the chromatographic system is adequate for analysis. To ascertain the effectiveness of the method developed in this study system suitability tests were performed on freshly prepared standard stock solutions of Paracetamol, Diclofenac Sodium and Chlorzoxazone.

CONCLUSION:

The proposed HPTLC method provides simple, accurate and reproducible, quantitative analysis for simultaneous determination of Paracetamol, Diclofenac sodium, Chlorzoxazone in tablet dosage form. The method was validated as per ICH guidelines. Statistical test indicates that the proposed HPTLC method reduced the duration of analysis and appears to be equally suitable for routine determination of Paracetamol, Diclofenac sodium, Chlorzoxazone simultaneously in pharmaceutical formulation.

ACKNOWLEDGEMENT:

The authors are thankful to Hetero Labs Ltd. Mumbai, India for supplying the gift samples and to Anchrom Labs Mumbai, India for providing technical support. We are also thankful to the Principal S.G.R.S College of Pharmacy,. Saswad, Pune for providing necessary facilities.

REFERENCES:

1. www.drugs.com

2. www.wikipedia.com.

3. Indian Drugs Review. A. Mediworld Publication; New Delhi, (2006).

4. Tripathi K. D.; Essential of Medical Pharmacology; Jaypee Brothers Medical Publishers (P) Ltd.; New Delhi, 5^th Ed., (2003), 48-52.

5. Indian Pharmacopoeia Govt. of India. Ministry of Health and Family Welfare. Controller of Publication, Vol. I, 244, (1996).

6. British Pharmacopoeia, Vol. III, HMSO, Cambridge, International edition, , 2257, 2544, (2003).

7. United States Pharmacopoeia, 26^th edition. US Pharmacopoeial Convention INC (NF-21), 18,441, (2004)

8. ICH Q2A Harmonized Tripartite Guideline, Text on Validation of Analytical Procedures, IFPMA In: Proceedings of International Conference of Harmonization, Geneva, March (1994).

9. ICH Q2B Harmonized Tripartite Guideline, Text on Validation of Analytical Procedures Methodology, Proceedings of International Conference of Harmonization, Geneva, March (1996).

10. ICH Guidance on Analytical Methods, International Convention on Quality for Pharmaceutical Industry, Toronto, Canada, September, (2002).

11. Reviewer guidance, Validation of Chromatographic methods,:7-29 (1994)

12. Sethi PD, High Performance Thin Layer Chromatography: Quantitative analysis of pharmaceutical formulations, CBS Publications, New Delhi,162-5, (1996)

Received on 04.07.2009 Modified on 09.08.2009

Asian J. Research Chem. 2(3): July-Sept., 2009, page 306-309