INTRODUCTION:

Diabetes is not a single disease but rather is a group of metabolic disease characterized by hyperglycemia caused by inadequate insulin secretion with or without a simultaneous decrease in hormone action at its receptor¹. Combination therapy has been shown to achieve greater blood glucose, lowering than monotherapy, because different classes have different and complimentary mechanism of action. The rapid introduction of combination therapy with two or three complementary oral antidiabetics helps in targeting the dual effect and also reduced adverse effect². Glimepiride is chemically 3-Ethyl-4-methyl-N-(4-[N-((1r, 4r)-4-methylcyclohexyl carbomyl) sulfamoyl] phenethyl)-2-oxo-2, 5-dihydro-1H Pyrrole-1-carboxamide, and it is an oral blood-glucose-lowering drug of the sulfonylurea class³. It lowers blood glucose by stimulating the release of insulin from pancreatic beta cells. Metformin chemically N, N-Dimethyl biguanide, acts by decreasing intestinal absorption of glucose reducing hepatic glucose production and increasing sensitivity⁴. The combination of sulfonyl urea with MET is largely used because both the drugs are ancient and large number of studies have demonstrated their synergistic effects.

Literature survey reveals that a few RP-HPLC methods are reported for the simultaneous estimation of GLI and MET^5-7. The main objective of this work was to develop a simple, rapid and sensitive RP-HPLC method for the simultaneous determination of GLI and MET and the method are validated as per ICH guidelines.

Figure 1.Chemical structures of (a) Metformin and (b) Glimepiride

EXPERIMENTAL:

Chemicals and Materials:

Metformin and glimepiride were obtained as gift sample from Micro Labs Ltd., Bangalore, India. HPLC grade acetonitrile, acetic acid and water were purchased from M/s. Merck Chemicals, Mumbai, India. Tablet formulation Gemer 2 (Sun Pharma, Sikkim, India, labeled to contain 500mg metformin and 2mg glimepiride) was procured from a local pharmacy.

Instrumentation and chromatographic conditions:

The HPLC system consisted of a Shimadzu LC-20AD pump, a Rheodyne 7725 sample injector with 20µl loop and a shimadzu SPD-20A diode array detector. The acquisition was performed by processing software “LC- solution” (Shimadzu Corp.) The method was developed on a phenomenex C₁₈ (250 ×4.6 mm, 5 µm) column maintained at ambient temperature. The mobile phase consists of acetonitrile and acetate buffer (pH 4) in the ratio 65:35 v/v delivered at a flow rate of 1.0 ml/minute. The injection volume was 20 µl and detection was carried out at a wavelength of 245 nm.

Preparation of standard solution and calibration curves:

Standard stock solutions of metformin and glimepiride were prepared separately by dissolving 50 mg of GLI and MET in 25 ml acetonitrile in 50 ml standard flasks. The resultant solutions were sonicated for 5 minutes and volume was made up to 50 ml with acetonitrile. From this 5 ml was transferred into 50 ml volumetric flask and made up to volume to get 100 μg/ml each of GLI and MET. Mixed standards containing 0.1 - 0.6 μg/ml of GLI and 25 - 125 μg/ml of MET were prepared by transferring appropriate aliquots from the above solutions and 20 μl of each concentration of the drug were injected in to HPLC system in triplicate and their chromatograms were recorded. Peak areas were recorded and standard calibration curve of area against concentration was plotted both for GLI and MET.

Preparation of sample stock solution:

Ten tablets of sample (Gemer 2) were accurately weighed. Their average weight was calculated, pulverized to fine powder. An amount equivalent to 500mg MET was accurately weighed and transferred to suitable 100 ml volumetric flask add 20ml of acetonitrile and the resultant solution was sonicated for 15 minutes. The volume was adjusted with acetonitrile and filtered through 0.45 µm nylon filters (Millipore, Milford, USA). From the above solution 100 µg/ml solution was prepared by using acetonitrile. From this solution 20 µl is injected into HPLC system and chromatogram were recorded.

RESULT AND DISCUSSION:

Method Validation:

Chromatography:

The non-polar nature of GLI necessitated the selection of C₁₈ reverse phase column. Initially reversed phase LC separation was tried using methanol: PO₄ buffer (pH 4, mixed) in the ratio of 70:30 was tried. Metformin was not eluted in this system. Then methanol: acetate buffer (pH 4) in the ratio 65:35 was tried and peak splitting was observed with MET. From the literature survey we concluded that acetonitrile is essential. By performing acetonitrile and different system we confirmed acetonitrile and acetate buffer (pH4) was optimized. Resolution, theoretical plate, symmetry of peaks and retention time were found to be satisfactory in this mobile phase composition. The retention time for GLI was found to be5.709±0.02 and MET 2.517±0.05 respectively (Fig 3).

System suitability:

System suitability parameters such as number of theoretical plates, retention time, tailing factor and resolution are determined. The results obtained are shown in Table1. The number of theoretical plates for GLI and MET were 9762 and 10521 respectively.

Figure 2: Typical chromatogram of standard (a) MET (Rt 2.517) and (b) GLI (Rt 5.709)

Table 1: Results of system suitability testing

Drug	Retention time (min)	Area	Tailing factor (T)	Theoretical plates (N)	Resolution (R)
GLI	5.709 ± 0.02	6617.34	0.21	9762	4.151
MET	2.517 ± 0.05	524806.78	0.19	10521	4.151

Figure 3: Sample chromatogram of (a) MET and (b) GLI

Linearity:

GLI and MET showed a linearity of response between 0.1-0.6µg/ml and 25-150µg/ml respectively. The peak area versus concentration data was performed by least square linear regression analysis whereby slope, intercept and correlation coefficient determined.

Table 2: Linearity parameters for calibration curve:

Parameter	GLI	MET
Retention time (R_t)	5.709 ± 0.02	2.517 ± 0.05
Linearity range (µg/mL)	1 - 10	2 – 20
Regression equation (y = mx+c)	y = 67246x - 179.3	y = 18015x + 55882
Correlation coefficient (r)	0.999	0.998
Limit of detection (µg/ml)	0.012	0.0125
Limit of quantification (µg/ml)	0.038	0.2211
Standard deviation of slope (S_a)	1.214	1.197
Standard deviation of intercept (S_b)	0.9811	1.026
Regression coefficient (r²)	0.999	0.998
Method precision (%RSD)	1.65	1.74

Sensitivity:

The LOD was determined based on standard deviation (SD) of response and slope (S) and it can be expressed as LOD=3.3 SD/S. The LOQ was determined as the lowest amount of analyte that was reproducibility quantified and expressed as LOQ=10 SD/ S and the results are reported in Table2.

Accuracy and Precision:

Accuracy and precision of the assay were determined using replicate analysis of quality control samples at three concentrations, by performing the complete analytical runs on the same day and also on three consecutive days. The data obtained for both GLI and MET were within the acceptable limits to meet the ICH guidelines. The results are depicted in Table 3 and 4 and Fig 4.

Robustness:

Robustness was determined by deliberately varying certain parameters like flow rate, pH of mobile phase, column temperature and change in detector wave length. For all changes in conditions that samples were analyzed in triplicate. The retention factor and resolution between GLI and MET were evaluated in each condition. Results of analysis are summarized in Table 5.

Application to solid dosage form:

The proposed method can be used for estimation of both drugs in solid dosage forms. The results are depicted in Table 6(a) and (b) indicate that each drug in tablet corresponds to requirement of label claim. The low %RSD value (<2%) confirmed the suitability of method for routine analysis of GLI and MET in pharmaceutical dosage forms.

Table 3: Accuracy of the proposed method

Drug	Level (%)	Theoretical concentration (µg/ml)	Observed concentration (µg/ml) ± SD^*	Mean recovery (%)	SEM	% RSD
GLI	50	0.15	0.149 ± 0.001	99.33	0.00058	0.671
	100	0.6	0.595 ± 0.0081	99.16	0.0047	1.361
	150	1.5	1.51 ± 0.0193	100.6	0.112	1.29
MET	50	37.5	37.61 ± 0.04	100.29	0.023	0.106
	100	150	149.83± 0.109	99.89	0.063	0.072
	150	375	376.01± 0.112	100.27	0.065	0.029

Mean of three replicates

Table 4: Precision study of the proposed method

Drug	Precision	Initial amount (µg/ml)	Amount found (µg/ml) ± SD^*	%Recovery	%RSD
GLI	Intraday	0.1	0.998±0.01	99.80	1.002
		0.4	0.401±0.005	100.25	1.246
		0.6	0.596±0.0059	99.33	0.989
	Interday	0.1	0.101±0.001	101.0	0.990
		0.4	0.394±0.003	98.50	0.076
		0.6	0.602±0.005	100.33	0.831
MET	Intraday	25	24.92±0.251	99.68	1.007
		75	75.01±0.329	100.01	0.439
		150	149.18±0.504	99.45	0.339
	Interday	25	25.01±0.211	100.04	0.844
		75	74.89±0.289	99.85	0.386
		150	149.56±0.651	99.71	0.435

^*Mean of three replicates

Table 5: Results of robustness study

Parameter	Rt		Resolution	%RSD
Parameter	GLI	MET	Resolution	GLI	MET
Change in pH of mobile phase
pH 3.6	5.69	2.52	4.752	0.413	0.672
pH 3.8	5.72	2.51	4.751	0.356	0.880
pH 4.2	5.70	2.51	4.752	0.948	0.874
Change in temperature
20º C	5.68	2.52	4.754	0.856	0.988
25º C	5.70	2.52	4.752	1.009	0.981
30º C	5.70	2.53	4.751	0.888	0.775
Change in flow rate
0.8 ml/min	5.73	2.54	4.752	1.213	0.985
1.2 ml/min	5.70	2.50	4.751	1.031	1.061
Change in wavelength
243nm	5.69	2.50	4.753	0.424	0.944
247nm	5.69	2.50	4.753	0.506	0.671

Table 6 (a): Data for assay of GLI and MET

Drugs	Area of Standard	Wt. of tablet (gm)	Area of sample	Content of tablet powder (gm)	Av. content (gm)
GLI	6658.56	1.160	6638.34	0.0019	0.0019
		1.161	6692.94	0.0018
		1.160	6700.38	0.00199
		1.160	6618.61	0.0019
		1.160	6667.17	0.0019
MET	524200.81	1.161	524100.07	0.4832	0.4834
		1.160	524090.11	0.4833
		1.161	524450.09	0.4837
		1.162	524125.01	0.4833
		1.160	524119.98	0.4833

Table 6 (b): Results from HPLC quantification of GLI and MET in tablets

Sample

Label claim (mg)

Amount present (mg)

SD^*

%RSD

GLI

MET

GLI

MET

GLI

MET

GLI

MET

Gemer*2

2.0

500

0.0019

0.4834

0.00002

0.004

1.053

0.827

*Mean of five replicate