Development And Validation of a Simple, LCMS Compatible New Assay Method for The Simultaneous Estimation of Anti-Diabetic Drugs Metformin, Sitagliptin and Glimepiride

Metformin hydrochloride (MET) is chemically 1, 1-dimethylbiguanide hydrochloride (Fig. 1 a.) is the main first-line medication for the treatment of type 2 diabetes, particularly in people who are obese. It is also used in the treatment of polycystic ovary syndrome. Similarly, Glimepiride (GLM) is chemically 1- [ [p- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-1 carboxamido) ethyl] phenyl] sulfonyl]-3- (trans-4-methylcyclohexyl) urea (Fig.1 b.) is an anti-diabetic medication used to treat type 2 diabetes. Glimepiride was patented in 1979 and approved for medical use in 1995. It is available as a generic medication and sitagliptin phosphate monohydrate (SIT) is (3R)-3-amino-1-[3-(trifluoromethyl)-5,6-dihydro-triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one phosphate onohydrate (Fig 1 c.) Sitagliptin is an anti-diabetic medication used to treat type 2 diabetes.  It is available in oral dosage form. It is also available in a fixed-dose combination as sitagliptin/metformin. Recently, Combination therapy in Type 2 diabetes is more efficacious and is better tolerated than high dose of Individual drug products. It offers convenient dosing and improves patient compliance in Diabetes treatment. The literature review reveals that the HPLC methods for the simultaneous estimation of Metformin, Glimepiride and Sitagliptin in tablet dosage are quite tedious in sample preparation, using complex buffers and longer run times. Hence the present study was aimed at developing simple, short, reliable and accurate RP- HPLC method for simultaneous estimation of Metformin, Glimepiride and Sitagliptin in tablet dosage form which can also be extended to the LCMS analysis for Metabolite identification.

Fig 1: a) Structure of Metformin Hydrochloride, b) Glimepiride and c) Sitagliptin phosphate

Materials and Methods:

The API Ref Standards of Metformin, Glimepiride and Sitagliptin phoosphate were provided as a gift sample FDC Ltd.    Mumbai, India. The solvents used were of following make: Acetonitrile, (HPLC grade Thermofisher), HPLC Water (MilliQ), Trifluoro acetic acid (AR grade Spectrochem), Methanol (HPLC grade Thermofisher), 0.45µm filter (Millipore, Bangalore). Glimepiride 2mg and Metformin HCl 1000mg combination Tablet & Sitagliptin 50mg and Metformin 1000mg tablet were procured from a local pharmacy. (Alkem Lab – Glimepiride and Metformin tablet 2mg / 1000mg; Sun pharma – Sitagliptin and Metformin tablet 50 mg / 1000 mg) Various C18 and C8 columns with different dimensions and carbon load were tried during development with different combination of Acetonitrile, Methanol and various volatile buffers and the best results were obtained on Hemochrom C8 column (4.6mm x 250mm; 5µm).

Chromatographic Conditions: 

The method development, validation and analysis of the drug was carried out Waters Alliance HPLC 2695 Separation module equipped with 2996   PDA Detector. The column used was Reverse Phase Hemochrom C8 column (4.6mm x 250mm; 5µ), ambient temperature at the flow rate of 1 ml/min using Empower software. Mobile phase used was Acetonitrile and 0.05% Aq. TFA in gradient mode.  The PDA detector was set at detection wavelength of 228 nm for Glimepiride and 210 nm for Sitagliptin and Metformin respectively.  The Standard and sample solutions for Metformin and Sitagliptin estimation were prepared in 0.05% TFA whereas Methanol was used as a diluent for Glimepiride.

Preparation of Standard Stock Solution: Preparation of Std. Metformin Solution (Stock A): An accurately weighed quantity, 50 mg of Metformin was taken in 50 ml Volumetric flask, about 25ml of Diluent was added and sonicated for 15 mins and diluted up to 50 ml to produce a solution of 1000 ppm. From the freshly prepared standard stock solution (1000 ppm), 100 ppm working standard solution was prepared by diluting 1 ml to 10 ml in a volumetric flask using the diluent

Preparation of Std. Sitagliptin Solution (Stock B): An accurately weighed quantity, 25 mg of Sitagliptin was taken in a 50 ml volumetric flask, about 25ml of Diluent was added and sonicated for 15 mins and diluted up to 50 ml to produce a solution of 500 ppm. From the freshly prepared standard stock solution (500 ppm), 1ml stock solution was pipetted out in 10 ml of a volumetric flask, the volume was made up to 10 ml with mobile phase to get a final concentration of 50 ppm.

Preparation of Std. Glimepiride Solution (Stock C): An accurately weighed quantity, 50 mg of Glimepiride was taken in a 50 ml volumetric flask. About 25ml of the Diluent was added, sonicated for 15 mins and diluted upto 50 ml to produce a solution of 1000 ppm. From the freshly prepared standard stock solution (1000 ppm), 1ml stock solution was pipetted out in 10 ml of a volumetric flask, and volume was made up to 10 ml with mobile phase to get a final concentration of 100 ppm.

Method Validation: The working standard of various concentrations was prepared by taking aliquots of respective Standard stock solutions and diluting to get the required concentration for injection to obtain a calibration plot  11

Assay Preparation for Commercial Formulation:

1) For estimation of Metformin (500 mg) and Sitagliptin (50 mg) from tablets:

Twenty tablets were finely powdered in a mortar and pestle. Sample powder equivalent to weight of one tablet was accurately weighed and transferred into a 500 ml volumetric flask. About 250 ml of the diluent was added and sonicated for 15 mins. And diluted up to 500 ml with the diluent (500 ppm). Further 2 ml of this solution was diluted to 10 ml with the diluent. (100ppm). Few ml of the 100 ppm solution was filtered through a PTFE 0.45 micron membrane filter paper and used for assay of Metformin. For Sitagliptin, 1 ml of the tablet solution was diluted to 10 ml (50 ppm) The representative chromatograms of test Metformin and Sitagliptin is shown in Fig. 2. The amounts of Metformin and Sitagliptin per tablet were calculated by from the calibration curve. The analysis procedure was repeated three times with tablet formulation. Tablet Assay for %Label claim for % RSD was calculated and the result are as shown in Table 1.

1) For estimation of Metformin (1000 mg) and Glimepiride (2 mg) from tablets:

Twenty tablets were finely powdered in a mortar and pestle. Sample powder equivalent to weight of one tablet was accurately weighed and transferred into a 1000 ml volumetric flask. About 500 ml of the diluent was added and sonicated for 15 mins. and diluted up to 1000 ml with the diluent (1000 ppm). Further 1 ml of this solution was diluted to 10 ml with the diluent. (100ppm). Few ml of the 100 ppm solution was filtered through a PTFE 0.45 micron membrane filter paper and used for assay of Metformin. For Glimepiride, weight equivalent to 1 tablet was taken in a 100 ml flask, about 25 ml Diluent Methanol was added, sonicated for 15 mins, and then diluted to 100 ml with Methanol (200 ppm). 5 ml of the above  tablet solution was further diluted to 10 ml (100 ppm) The representative chromatogram of test Metformin and Glimepiride is shown in Fig. 3. The amounts of Metformin and Glimepiride per tablet were calculated by from the calibration curve. The analysis procedure was repeated three times with tablet formulation. Tablet Assay for %Label claim result was shown in Table 1.

Fig 2: Representative Chromatogram of Metformin Sitagliptin Formulation

Fig 3: Representative Chromatogram of Glimepiride Metformin Formulation

RESULTS:

Linearity and Range: The data obtained in the calibration experiments when subjected to linear regression analysis showed a linear relationship between peak areas and concentrations in the range 50 -150 µg/ml for Metformin & Glimepiride and 25-75µg/ml for Sitagliptin. The respective linear equation for Metformin was y = 43392x + 142874, Glimepiride equation y = 29092x + 26589 and Sitagliptin equation y = 8478.2x + 13608, where x is the concentration and y is area of peak. The correlation coefficient was 0.999. The calibration curve of Metformin, Glimepiride and Sitagliptin is depicted in Fig. 4, 5 and 6 respectively.

Fig 4: Calibration Curve of Glimepiride

Fig 5: Calibration Curve of Metformin

Fig 6: Calibration Curve of Sitagliptin