INTRODUCTION:

Cefixime is 8-[[2-(2-Amino-1, 3-thiazol-4-yl)-2-(carboxymethoxyimino) acetyl] amino]-4-ethenyl-7-oxo-2-thia-6-azabicyclo [4.2.0] oct-4-ene-5-carboxylic acid. It is an oral third generation cephalosporin antibiotic. It is used to treat gonorrhea, tonsilitis, and pharyngitis. ^1,2,6 Cefixime kills bacteria by interfering in the synthesis of the bacterial cell wall, .it’s mode of action and spectrum of activity is more or less similar to other cephalosporin’s of it’s own class.³

Cloxacillin is (2S, 5R, 6R)-6-{[3-(2-chlorophenyl)-5-methyl-oxazole-4-carbonyl] amino}-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid.^1,2,6 Cloxacillin is a semisynthetic antibiotic in the same class as penicillin. It is sold under a number of trade names, including Cloxapen and Orbenin. Cloxacillin is used against staphylococci that produce beta-lactamase. ³

These drugs are official in IP, BP and USP. HPLC method has been reported for this combination. ^[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. ^{4, 5}

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 ahs has been developed to check whether it is suitable for different conditions and with varying concentrations of the sample. ^{10, 11}

The aim of the present work is to develop and validate HPTLC method for estimation of Cefixime and Cloxacillin in bulk and tablet dosage form.

MATERIALS AND METHODS:

· Instrumentation:

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

· Materials and reagents:

Analytically pure gift samples of Cefixime and Cloxacillin were obtained from Hetero Labs Ltd. Mumbai; India used as working standards. While, n-butanol, Methanol Water and Formic acid of HPLC grade (Obtained from Merck Chem.) to prepare mobile phase were used without further purification.

· Standard stock solutions:

Standard stock solutions containing 0.5mg/ml of Cefixime and 1mg/ml of Cloxacillin were prepared by dissolving 5mg and 10mg of respective standards of Cefixime and Cloxacillin in 10ml methanol in 2 different flasks and are used as working standard solutions.

· Sample preparation:

Twenty Zifi X 200 tablets manufactured by FDC containing 200mg of Cefixime and 500mg of Cloxacillin were weighed and powdered. An amount of powder equivalent to 200mg of Cefixime and 500mg of Cloxacillin of was transferred to 250ml calibrated volumetric flask containing 100ml methanol sonicated for 15min. Then the resulting solution was filtered using 0.45µm filter. Take 0.5ml of solution from filtrate and dilute it for 10ml of methanol. Then take 5ml of this solution and again dilute it for 10ml of methanol. A sample solution (9µl containing 4500ng of Cefixime and 6µl containing 6000ng of Cloxacillin) was spotted for assay.

· 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 15s µL-1 by means of a Camag (Muttenz; Switzerland) Linomat V sample applicator equipped with a 100-µL syringe (Hamilton). The mobile phase selection was done on the basis of “Trial and Error” on Bulk drug.¹² The distance between the bands was 10.0mm. Ascending development of the plate, migration distance 80mm was performed at 25±2⁰C with n-Butanol: Methanol: Water: Formic acid (8:6:4:0.3v/v/v/v) as mobile phase in a Camag twin trough chamber previously saturated for 20min with paper. The average development time was 20min. UV 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.The scanning showed the maximum absorbance of Cefixime and cloxacillin at 293nm and 343 nm respectively.

RESULT AND DISCUSSION:

Validation of HPTLC method ^8-12

· Linearity:

Amounts of standard solutions equivalent to 1.5-9 µg/spot of Cefixime and 2-1.2µg/spot of cloxacillin 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 Cefixime and cloxacillin was assessed in the concentration ranges 1.5-9 µg/spot of Cefixime and 2-1.2µg/spot of cloxacillin, in terms of intercept and correlation coefficient values. The calibration spots showed the correlation coefficients (r=0.9956, 0.9988, 0.9988), intercept were (239, -7.8) respectively over the concentration range studied with six replicate readings of each concentration.

· Sensitivity:

The sensitivity of measurement of Cefixime and Cloxacillin 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 curve¹². The results of sensitivity are as shown in Table No. 1

Table No. 1: Results of assay of Cefixime and Cloxacillin in pharmaceutical dosage forms

Brand	Sr. No.	Amount Label claimed
Brand	Sr. No.	Cefixime (200mg)	Cloxacillin (500mg)
Zifi-X-200	1	186.45	445.34
	2	187.90	446.73
	3	186.34	445.62
	4	186.74	445.73
	5	186.66	446.31
Mean assay		186.18	445.94
R.S.D (%)		1.54	1,87
LOD (ng/spot)		50	20
LOQ (ng/spot)		150	60

· Assay and Precision evaluation:

The amount of Cefixime and Cloxacillin was applied in number of replicates of both pharmaceutical preparations (n = 6) and the assay results are reported in Table No. 2. Precision studies were performed using standard solution of Cefixime and Cloxacillin 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 Cefixime and Cloxacillin standard drug solution within the calibration range, three times on the same day. Inter-day precision (% CV) was assessed by analysing Cefixime and Cloxacillin 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.

Table No. 2: Results from precision evaluation

Drug	Intra-day precision			Interday precision
Drug	SD	% CV	SE	SD	% CV	SE
Cefixime	1.94	1.65	0.79	2.24	1.85	1.02
Cloxacillin	1.87	1.21	0.71	2.18	1.34	0.88

· 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 Cefixime and 4000ng/spot of Cloxacillin was prepared from tablet solution and spiked with amounts equivalent to 50, 100. 150 % of amounts of drugs in the original solution. When these solutions were analyzed the recoveries were found to be within the limits of 89-93%. The reports of recoveries are as shown in the Table No.3

Table No. 3: Results from recovery studies

Component	Label claim (mg/ tablet)	Initial amount (µg)	Amount added (µg)	Amount recovered (µg)	% recovered	% RSD
Cefi xime	200	3	1.5	1.49	93.73	1.65
		3	0	2.81	93.76	1.45
		3	4.5	4.18	93.04	1.63
Cloxa cillin	500	4	2	1.79	89.70	1.21
		4	0	3.59	89.87	1.72
		4	6	5.39	89.98	1.19

· Specificity:

The mobile phase designed for the method resolved the drugs very efficiently as shown in Figure No.1. The R_f values of Cefixime and Cloxacillin were found to be 0.28 and 0.45 respectively. The peak purity was tested by correlating the spectra’s of Cefixime and Cloxacillin at the peak start (S), peak apex (A) and peak end (E) positions. Correlation between these spectra’s indicated purity of Cefixime and Cloxacillin peak correlation r (S, A) = 0.9996, 0.9994 and r (A, E) = 0.9997, 0.9992. It can be thus concluded that no impurities or degradation products were found with the peaks of standard solution.

Figure No.. 1: A typical chromatogram of Cefixime and Cloxacillin measured at 293nm and 343nm respectively. Mobile phase- n-Butanol: Methanol: Water: Formic acid (8:6:4:0.3v/v/v).

· 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 Cefixime and Cloxacillin

CONCLUSION:

The proposed HPTLC method provides simple, accurate and reproducible, quantitative analysis for simultaneous determination of Cefixime and Cloxacillin 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 Cefixime and Cloxacillin simultaneously in pharmaceutical formulation

ACKNOWLEDGEMENT:

The authors are thankful to BCUD University of Pune for funding the project, 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. http//www.drugs.com

2. http//www.wikipedia.com.

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

4. Indian Pharmacopoeia (1996) Govt. of India. Ministry of Health and Family Welfare. Controller of Publication, Vol. I and II, 1996, pp. 244, 556.

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

6. Drugs Today (DT43), Jan-March 2004 pp.174.

7. G. Rathinavel, “A Validated RP-HPLC Method for Simultaneous Estimation of Cefixime and Cloxacillin in Tablets”, E-Journal of Chemistry, Vol.5 (3), July 2008, pp- 648-651.

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 1994, pp 7-29

12. Sethi PD, High Performance Thin Layer Chromatography: Quantitative analysis of pharmaceutical formulations, CBS Publications, New Delhi, 1996 pp 62-5.

Received on 12.09.2009 Modified on 09.11.2009

Asian J. Research Chem. 3(2): April- June 2010; Page 299-301