INTRODUCTION:

Metformin chemically is 1,1-Dimethylbiguanide hydrochloride¹.Metformin is an oral hypoglycaemic agent, clinically used in the treatment of diabetes patients which facilitates glucose uptake level in skeletal muscle and reduces the free fatty acid oxidation⁵ .Metformin is official in IP², EP³, BP⁴. Fenofibrate chemically is 1-methyethyl 2-[4-(chlorobenzoyl)phenoxy]-2-methyl- propanoate, is a febric acid derivative ,which activates peroxisome proliferators-activation receptors and reduces the total plasma cholesterol , it is used for regulating plasma lipids and treatment of hyperlipoproteinaemias⁶. Fenofibrate is also official in BP¹⁴. Results from a clinical trial showed that combination therapy of 500 mg of MET and 80 mg FNB significantly reduced LDL cholesterol levels when compared to either treatment alone. Coadministration of MET and FNB to provide a pharmaceutical composition containing both active components, metformin and a fenofibrate, whilst maintaining the bioavailability of each of the two components equivalent to or superior to that obtained with metformin alone or with the fibrate alone^7-8. The synthetic mixture of combination of both the drug were prepared in the ratio of 500: 80 (MET:FNB) including excipients required for tablet dosage form.

Various methods such as, HPLC^9-10, HPTLC¹¹, UV^12-13 spectrophotometrymethod have been reported for individual drugs in formulation. Literature survey reveals that no method has been reported for simultaneous analysis of MET and FNB in its combination. In the present investigation, two simple, rapid and accurate methods have been developed for simultaneous estimation of MET and FNB from their synthetic mixture.

MATERIAL AND METHODS:

A Shimadzu UV/Visible double beam spectrophotometer model 1700 was employed with a pair of 10mm quartz cells. Methanol AR grade was used as solvent procured from Universal Laboratories limited, Mumbai. Tablet containing MET 500mg and FNB 80mg were prepared inhouse by using common excipents like microcrystalline Cellulose as a filler, 5% Polyvinylpyrrolidone in ethanol as a binding agent and Magnesium stearate as a lubricant by wet granulation technique.

Fig. 1: Overlain spectra of MET and FNB

Table 1: Assay and Recovery studies of MET and FNB in synthetic mixture

Method	Label Claim (µg/tab)		*Amount Found (%)		Standard Deviation*		%Mean Recovery*
	MET	FNB	MET	FNB	MET	FNB	MET			FNB
	MET	FNB	MET	FNB	MET	FNB	80	100	120	80	100	120
A	500	80	100.64	99.92	0.3465	0.2372	99.42	100.54	99.82	99.03	100.07	98.96
B	500	80	99.93	99.63	0.6186	0.6072	100.70	100.15	100.16	99.89	99.90	100.09

A - Simultaneous equation: B –Multicomponant analysis: MET and FNB – Metformin hydrochloride and Fenofibrate respectively.* denotes n = 6, average of six estimations.

Fig. 2: Multicomponent Analysis of mixed standards

PROCEDURE:

Standard stock solutions having 100 µg/ml of MET and FNB were prepared by dissolving separately 10 mg of each drug in 100 ml methanol.

For the simultaneous equation method, appropriate dilutions of two drugs were prepared separately using standard stock solutions and the same were scanned in the range of 400 nm to 200 nm to obtain overlain spectra. The absorbance maximum observed for MET and FNB at 237.5 nm and at 287.5 nm respectively. However though fenofibrate showed good linearity at its wavelength (λ_max) but Metformin did not reproduce good linearity and hence λ_max selected was 266.0 nm. Various dilutions were made of two drugs individually and same were scanned in the range of 400 nm to 200 nm. MET and FNB showed linearity in the range 5-30 µg/ml and 0.8-4.8 µg/ml at their respective selected wavelengths. The absorptivity coefficients of each of the two drugs were calculated at 237.5nm and 266.0 nm. A set of two simultaneous equations using the absorptivity coefficient values

A1= 87.54 × C₁ + 3.03× C_{2 …} (I)

A₂= 19.34 × C₁ + 35.42 × C_{2 …...}(II)

Where, C₁ and C₂ are concentrations of MET and FNB respectively in g/l in the sample solution. A₁ and A₂ are absorbances of sample solution measured at 237.5 nm and at 266.0 nm of MET and FNB, respectively, while 87.54 and 19.34 are the absorptivity values at 237.5 nm of MET and FNB respectively, and 3.03 and 35.42 are the absorptivity values at 266.0 nm of MET and FNB respectively. The absorbances were recorded at selected wavelengths 237.5 nm and 266.0 nm and the amount of drug present in the mix standard solution were obtained by using the above equation.

In the second method, multicomponent mode of the instrument was used which has inbuilt software to calculate the concentration of the individual component in a mixture. The sampling wavelengths selected for the estimation of MET and FNB were 237.5 nm and 266.0 nm respectively. The concentrations of mixed standard solution were entered in multicomponent mode. The absorbance spectra of mix standard and sample solutions were measured at selected wavelength. The instrument processes the data and gives individual concentration of the two drugs present in sample solution directly.

The synthetic mixture of combination of both drugs were prepared in 500:80(MET: FNB) ratio including excipients required for tablet dosage form. From this mixture amount equivalent to 25 mg of MET and 4 mg FNB were taken and diluted up to 50 ml. The solution was kept for sonication for about 30 min. The solution was then filtered through Whatman filter paper No.41 to it 6 mg of pure std. FNB was externally added (as external addition method). From this solution 25:10mg/ml (MET: FNB) was prepared and scanned using proposed methods and their results were obtained. Accuracy of the analysis was determined by performing recovery studies of MET and FNB at 80%, 100% and 120% level as per ICH guidelines¹⁵.

RESULT AND DISCUSSION:

The proposed methods for simultaneous estimation of MET and FNB in sample solutions were found to be simple, accurate and reproducible. The values of standard deviation were satisfactory and recovery was close to 100% indicating

reproducibility and accuracy of the methods (Table 1). The overlain spectra of MET and FNB and multicomponent analysis of mixture shown in Fig.1 and 2 respectively. The LOD and LOQ were found to be 0.2054µg/ml and 0.6224µg/ml for MET and for FNB 0.0161 µg/ml and 0.0490 µg/ml respectively.

ACKNOWLEDGEMENTS:

Authors are thankful to Concept Pharmaceuticals Ltd., Aurangabad for gift sample of Fenofibrate and also to Emcure Pharmaceuticals Ltd.,( R and D) Bhosari, Pune for gift sample of Metformin hydrochloride to carry out this work.

REFERENCES:

1. Budawari S, Eds, In; The Merck Index, 13th Edn., Merck & Co., Inc., Whitehouse Station, NJ, 2003. 1016,1335,3949.4002.

2. Indian Pharmacopoeia, New Delhi.1996(I).469

3. European Pharmacopoeia, 4^th edition. 2003.1246

4. British Pharmacopoeia, 2004 (II). 805,1268.

5. Waller DG et al. Medical Pharmacology and Therapeutics,2^nd edition: 457-470.

6. Kothapalli Lata et al. Development and validation of spectrophotometric method for simultaneous estimation of

atrovastatin calcium and Fenofibrate I pharmaceutical formulation. J.Pharma.Research. 2007; 6(3):145-147.

7. Dawson et al. Pharmaceutical formulations comprising metformin and a fibrate, and processes for their obtention. 2005. http://www.freepatentsonline.com/EP1559419A1.html.

8. Dawson et al. Pharmaceutical Formulations Comprising Metformin And A Fibrate,And Processes For Obtaining Them.2008. http://www.freepatentsonline.com/US20080020046A1.html.

9. ShenHR, LiZD and Zhong MK.Pharmazie.2006; 61:18

10. Rajeshwari KR et al.Indian J Pharm sci.2006;68:275

11. Gupta KR,Wankhede SB,and Wadodkar SV. Indian J Pharm Sci.2005;25:762.

12. Miaztal G and omsta L. J AOAC Int. 2005;82:479

13. Shahu R and Patel VB. Indian Drugs.2006;43:160.

14. British Pharmacopoeia, 2008 (I).891.

15. ICH,Q2A, text on validation of analytical procedures, International Conference on Harmonization, Geneva,1994 ; 1-8.

16. Martindale, In; The Complete Drug Reference, 32nd Edn., Pharmaceutical Press,589.

17. Yardimci C et al.,Talanta . 2007 ; 72 : 1416-1422.

18. Hassan SSM et al. Analytica Cimica Acta .1999 ; 378 : 299-311.

19. Bhavesh D et al. Indian J.Pharm.Res. Apr-Jun,2007 ;135-139.

20. Rang HP, Dale MM ,Ritter JM, Moore PK.5^thedition, Pharmacology,306-314.

21. Saad SM Hassan et al. Analytica Chemica Acta. 1999;378(1-3):299-311.

22. Aruna A.,Nancey K.,Indian Drugs.2000;37(11);593-536.

23. Puranik M et al. Indian Drugs.2005 ;42(7) :428-430.

Received on 15.03.2008 Modified on 20.03.2008

Research J. Pharm. and Tech. 1(2): April-June. 2008;Page86-88