INTRODUCTION:

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder with two major biochemical defects namely impaired insulin secretion and impaired insulin action at periphery. Resistance to insulin with eventual beta cell dysfunction is the key attributed to (T2DM), it us also characterized by resistance to insulin-stimulated glucose uptake in most of the patients Metformin hydrochloride (MET) is an oral antidiabetic drug and is chemically N,N-dimethyl imidodicarbonimidic diamide. Few UV Spectrophotometric methods^1,
2 , HPLC ^{3,4 ,5 ,6 ,7} and ion-pair HPLC⁸ method have been reported for the estimation of MET. Pioglitazone hydrochloride (PIO) is a member of type-2 oral antidiabetic agents called thiazolidinediones or insulin sensitizers, which makes body more sensitive to insulin. Chemically PIO is (±)-5-[4-[2-(5-ethyl-2-pyridinyl) ethoxy]phenyl]methyl]-2,4-thiazolidinedione^[1] . PIO is not yet official in any of the pharmacopoeia but MET is official in IP⁹, BP¹⁰ and USPNF¹¹ . Literature indicated an RPHPLC method and a MEKC method, for the determination of PIO from plasma as well as pharmaceutical preparations.

Glimepiride (GLP) chemically it is [[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyyroline-1- oxamide) ethyl] phenyl] sulfonyl]-3-(trans-4-ethylcyclohexyl) urea, it inhibits hepatic gluconeogenesis and is given as the first line monotherapy. (Yao et al., 2006; Jedlickaet al., 2004 and Kolte et al., 2004) were reported method for estimation of glimepiride in human plasma. (Chakradhar et al., 2007; Saleem et al., 2004) was published HPLC method for estimation of glimepiride and related substances (Deep et al., 2005; Kovarikova e tal., 2004; Khan et al., 2005) were also reported in the literature. Even though various methods were reported in the literature for estimation of glimepiride, Metformin and pioglitazone individually or in combination with other drugs, no method had been reported for simultaneous estimation of these three drugs using UV spectrophotoscopy in pharmaceutical dosage forms. Therefore, it was thought worthwhile to develop a simple, precise, accurate UV spectrophtometric methods for the simultaneous estimation of MET , PIO and GLP in combined tablet dosage form. The methods was validated according to the ICH (Q2A 1995) guidelines¹².

MATERIALS AND METHODS:

All spectrophotometric measurements were carried out using a Shimadzu uv/vis spectrophotometer 1700 series with a 1cm matched cell. An ultrasonicator was used for proper dissolution of the samples. Methanol (analytical grade) SD fine chemicals Mumbai was used as a solvent after considering the solubility factors of three drugs as well as the interference due to excipient matrix present in tablet formulation. Reference standards of piogilitazone hydrochloride, metformin and glimiperide were procured from micro labs pvt ltd Bangalore India.

Method A .Vierodt’s Simultaneous equation method.^13-14

This method is based on the absorption of drugs (X, Y and Z) at wavelength maxima of the other. The quantification analyses of GLP, MET and PIO in a ternary mixture were performed with the following equations:

A₁= ax₁bCx + ay₁bCy + az₁bCz ------ (Eqn 1)

A₂ = ax₂bCx + ay₂bCy + az₂bCz ------ (Eqn 2)

A₃ = ax₃bCx + ay₃bCy + az₃bCz ------ (Eqn 3)

Where, (b = 1) C x ,Cy and C z are the concentrations of GLP, MET and PIO respectively. A₁,A₂, and A₃ are absorbance of a mixture at 228nm,237nm and 267nm respectively. ax₁ ax_2,and ax₃are the absorptivity values of GLP,MET and PIO at 228nm,237nm and 267nm respectively. To calculate concentration of Cx consider equation (1) and (2) rearranging these two we get Cx = [A₂(ay₁₊az₁) – A₁(ay_{2 +} az₂)/ ax₂(ay₁₊az₁) – ax₁(ay_{2 +} az₂)].Similarly Cy = [A₁(ax₁₊az₁) – A₂(ax_{2 +} az₂)/ ay₂(ax₁₊az₁) – ay₁(ax_{2 +} az₂)]. Substitute the value of Cx and Cy in equation (3) we get the concentration of Z.

Method B. Q- Analysis (absorbance ratio method)¹⁵

This is the modification of simultaneous equation method. In the quantitative assay of multi components in admixture by the absorbance ratio method , absorbance is measured at two wavelengths. One being the λ _max of one component λ₂ and the other being a wavelength of equal absorptivity of the three components λ₁, ie., an iso-absorptive point (Pernarowski et al,1961). The concentrations of three drugs in an admixture can be calculated using equations

C _x = [Q _m – (Q _y + Q _z) x A₁/ Q _x – (Q _y + Q _z ) x ax₁] ,

C_y =[Q _m –(Q _x + Q _z) x A₁/ Q _y – (Q _x + Q _z ) x ay₁] ,

Q _z = A₁/az₁ – (C _x + C_y).

Where, C x ,Cy and Cz are the concentrations of GLP, MET and PIO respectively. A₂ is the absorbance of a mixture at λ₂237nm (λ _maxof MET), and A₁ is the absorbance of mixture at λ₁258 nm (iso-absorptive point ) Q m is the ratio of A₁/A₂. Q x, Q y and Q z are the ratio of ax₂/ax₁, ay₂/ay₁ and az₂/az_1. ax₂and ax₁are the absortivity coefficient of GLP at λ₂and λ₁, ay₂and ay₁are the absorptivity coefficient of MET at λ₂and λ₁, az₂and az₁ are the absortivity coefficient of PIO at λ₂and λ₁.

Analytical procedure:

Preparation of standard stock solution. The standard stock solution was prepared by dissolving10mg of each drug in 100ml volumetric flask separately using Methanol. Final working standard solution of 40µg/ml of GLP, MET and PIO were prepared by diluting 20ml of the above solution in 50ml volumetric flask with methanol. Working standard solution were scanned in the entire uv range of 200-400nm to determined the λ _max of three drugs. The λ _max GLP, MET and PIO were found to be 228nm, 237nm and 267nm respectively. Fig 1. Eight standard solution having concentration 2,4,6,8,10,12,16 and 20 µg/ml each drug were prepared in methanol using working standard solution. The absorbance resulting solutions was measured at 228nm,237nm and 267nm and calibration curve were plotted at these wavelengths and absorptivity coefficient was determined using calibration curve equation. Three simultaneous equations were formed using these absorptivity values. Similarly for Method 2 eight standard solution having concentration 2,4,6,8,10,12,16 and 20 µg/ml each drug were prepared in methanol using working standard solution and the absorbance at 257nm (iso-absorptive point), and at 237nm (λ _maxof MET) was measured and absorptivity coefficient were calculated using calibration curve.

Fig 1. Overlain spectra of GLP at 228nm,MET at 237nm, PIO at 267nm and iso absorptive point at 257nm.

Analysis of tablet formulation:

Twenty tablets (Brand name DAORIDE-PM Manufactured by Azine Health care pvt ltd Ahemdabad 22) were taken and their average weight was determined. They were crushed to fine powder containing the equivalent of 10mg of GLP, MET, and PIO was transferred in a 100ml volumetric flask. The GLP present in this tablet powder was 2mg which could not be found out accurately due to low absorbance. Hence to increase the accuracy, accurately weighed 8mg of pure drug of GLP was added to the crushed tablet which increased the amount of GLP to 10mg , it was then dissolved in methanol by intermittent shaking for 4-5 minutes. The volume was made up to 100ml and solution was filtered through whatman filter paper (No 41). The filtrate was further diluted to get final concentration of 10µg/ml.

For method A, the absorbances of sample solution , ie., A₁,A₂, and A₃,were recorded at 228nm,237nm and 267nm respectively, and concentration of three drugs in sample were determined using the equations.

Formulation	Ingredients in μg/ml	Method A			Method B
Formulation	Ingredients in μg/ml	*Amount found in μg/ml**	% Label claim	± SD	*Amount found in μg/ml**	% Label claim	± SD
DAORID-PM	GLP-10	10.036	100.094	0.096	10.018	100.040	0.388
	MET-10	10.002	100.068	1.474	9.994	99.938	0.941
	PIO-10	10.012	100.264	0.943	10.072	100.03	0.659

Table 1: Results of analysis of commercial formulation

*Average five determinations

Table 2. Optical characteristic and precision data.

Parameters	Method A							Method B
	228nm			237nm			267nm	257nm (iso absorptive point)
	GLP	MET	PIO	GLP	MET	PIO	PIO	GLP	MET	PIO
Beer’s law limit in μg/ml	2-20	2-20	2-20	2-20	2-20	2-20	2-20	2-20	2-20	2-20
Absortivity	70	50	42	32	35	30	80	26	26	26
(Є) L/mol/cm	3.4343 x 10³	8.2815 x10³	1.65018 x10³	1.56998 x10³	5.79705 x10³	1.1787 x10³	3.1432 x10³	1.27634 x10³	4.3063 x10³	1.02154 x10³
Regression equation (Y)*
Slope (b)	0.0097	0.0143	0.0241	0.335	0.817	0.766	0.0150	0.0020	0.0060	0.0312
Intercept (a)	0.1958	0.1753	0.1315	0.1011	0.6557	0.6777	0.0073	0.0110	0.0265	0.0084
LOD μg/ml	0.0421	0.0762	0.0512	0.0362	0.0383	0.0310	0.0162	0.0534	0.0516	0.0454
LOQ μg/ml	0.0287	0.3007	0.0531	0.0920	0.0663	0.0921	0.0162	0.0210	0.0361	0.0651
CV	0.2289	0.4466	0.2514	0.2821	0.2915	0.4747	0.2305	0.5563	0.6432	0.4382
%RSD(n=5)	1.64	1.32	1.46	1.73	1.83	1.91	1.23	1.45	1.23	1.28

Table 3: Results of recovery studies of commercial formulation

Formulation	Ingredients in μg/ml	Method A			Method B
Formulation	Ingredients in μg/ml	Amount of pure drug added in μg/ml	*Amount of drug found in μg/ml**	% recovery	Amount of pure drug added in μg/ml	*Amount of drug found in μg/ml**	% recovery
DAORID-PM	GLP-10	5	14.96	99.33	5	5.10	102.09
	MET-10	10	20.19	101.47	10	19.95	99.68
	PIO-10	15	25.30	101.69	25	25.13	100.63

*Average five determinations

A₁= (70)bCx + (50)bCy + (42)bCz ------ (Eqn 1),

A₂ = (32)bCx + (35)bCy + (30)bCz ------ (Eqn 2),

A₃ = (0) bCx + (0) bCy + (80) bCz ------ (Eqn 3).

To calculate the concentration of Z (PIO) equation (3) was considered. As GLP and PIO shows no absorbance at 267nm, concentration of Z was calculated. Concentration of X (GLP) was calculated by considering equation (1) and (2) and rearranging these two equations we got Cx = [(A₂(50 + 42) – A₁ (35 + 30)/ 32 (50 + 42)-70 (35 + 30)].To calculate the concentration of Y (MET) we Substituted the values of Cx and Cz in equation (2) we got the concentration of Y (MET).

For method B, the absorbances of sample solution, i.e. ., A₁, and A₂, were recorded at 257nm (isoabsorptive point) and 237nm. The concentration of three drugs in sample were determined using the equations. C _x = [Q _m – (Q _y + Q _z) x A₁/ Q _x – (Q _y + Q _z) x ax₁], C_y = [Q _m – (Q _x + Q _z) x A₁/ Q _y – (Q _x + Q _z) x ay₁], Q _z = A₁/az₁ – (C _x + C_y).The analysis procedure was repeated five times with tablet formulation for both the methods. The result of analysis of tablet formulation is shown in (Table 1).

RESULTS AND DISCUSSION:

In the proposed work two methods namely simultaneous equation (vierodt’s method) and Q-analysis (absorbance ratio method) were developed for simultaneous spectrophotometric analysis of GLP,MET and PIO in commercially available tablet dosage form using methanol as a solvent. In simultaneous equation method (method A) three wave lengths of respective absorbance maxima ie,. 228nm for GLP, 237nm for MET, and 267nm for PIO were used for the analysis of drugs. The λ _max selected for three components are reasonably dissimilar in addition to this three components do not interact chemically, thereby negating the initial assumption that the total absorbance is the sum of the individual absorbance. In absorption ratio method (method B) the two wave lengths selected for analysis of both drugs were 257nm (iso-absorptive point), and at 237nm (λ _maxof MET). The optical characteristic such as absorption maxima, Beer’s law limit, and molar absorptivities at selected wavelengths, the regression analysis using the method of least square was made for slope(m), intercept(b) and correlation (r) obtained from different concentrations were found to satisfactory. The reproducibility and precision of the methods were confirmed by % CV (Coefficient of Variance) values which are less than 2% and %RSD (Relative Standard Deviation)were found to be within the limit indicating good precision and reproducibility of the proposed methods. The results validation parameters are summarized in (Table 2). To study the accuracy of the proposed methods recovery studies were carried out by addition of known amount of standard drug solution of GLP, MET and PIO to pre analyzed sample solution. The resulting solution was analyzed by the proposed methods. The mean percentage recovery were found to be 99.33,101.47 and 101.69 %for GLP,MET and PIO respectively for method A and 102.09,99.68 and 100.63 % for GLP,MET and PIO respectively for method B indicates non interferences from the formulation exepients. The results for recovery studies were mentioned in (Table 3).

CONCLUSION:

Based on the validation data, it can be concluded that the proposed methods were accurate, precise and simple. No interference was found from excipients used in tablet formulation and hence methods are suitable for routine quality control analysis work.

ACKNOWLEDGEMENT:

The authors thank Prof. T. V. Narayana chairman Dr HLT college of pharmacy Kengel Channapatana; and Prof. R. Ramesh Principal, for the necessary facilities and encouragement.

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Received on 21.11.2009 Modified on 21.01.2010

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 518-521