Development and Validation of a Method for Simultaneous Determination of Metformin Hydrochloride and Sitagliptin Phosphate in a Formulation by RP-HPLC

Present study was aimed to develop and validate a reverse-phase high-performance liquid chromatography method for simultaneous determination of sitagliptin phosphate and metformin hydrochloride in a marketed formulation. The drug separation was performed on Hibar-240, Lichrosphere-100 C18 ODS (250 × 4.6 mm, 5 μm) column, at a flow rate of 1 mL/min. The mobile phase used was a mixture of methanol: potassium di-hydrogen phosphate buffer at a ratio of 70:30 v/v. The detection was carried out at a wavelength of 266 nm. The retention times of sitagliptin phosphate and metformin hydrochloride were found as 6.1 and 4.9 min respectively. Linear calibration curves with good correlation coefficients were obtained over the concentration ranges of 10 50 μg/mL for sitagliptin and 20 100 μg/mL for metformin. The limit of detection was 0.016 and 0.14 μg/mL and the limit of quantification was 0.048 and 0.42 μg/mL for sitagliptin phosphate and metformin hydrochloride respectively. Validation of the method demonstrated system selectivity, specificity, linearity, accuracy and precision. The developed method was found useful in the simultaneous analysis of sitagliptin phosphate and metformin hydrochloride in formulation.

Recently, the combination of sitaglipin and metformin has been recommended for use in the treatment of diabetes mellitus to improve glycemic control [8].Already few methods were reported for the determination of sitagliptin in pharmaceutical formulations or biological samples by spectrophotometry and HPLC [5].Analytical methods for determination of metformin include normal phase chromatography (silica and cyano), cation exchange chromatography, ion pair chromatography and reversed phase chromatography with UV or mass spectrometric detection [9].
The present work presents development and validation of a new and simple RP-HPLC method for simultaneous determination of metformin hydrochloride and sitagliptin phosphate in a formulation.

Materials and Reagents
HPLC grade acetonitrile and methanol were purchased from Sigma Aldrich (Bangalore, India).Analytical grade solvents namely Butanol, Methanol, Chloroform, Diethyl amine, Potassium dihydrogen phosphate and Formic acid were purchased from Sigma Aldrich (Bangalore, India).Pharmaceutical grade sitagliptin was a gift sample from Merck Sharp & Dohme (Cairo, Egypt).Metfromin was generous gift sample obtained from Aurobindo Pharmaceuticals Pvt Ltd. (Hyderabad, India).Janumet ® tablets nominally containing 50 mg of sitagliptin phosphate monohydrate and 500 mg of metformin per tablet were all kindly supplied by Merck Sharp and Dohme Co. (Cairo, Egypt).
The mobile phase was a mixture of methanol and potassium di-hydrogen phosphate buffer taken at a ratio of 70:30 v/v, delivered at a flow rate of 1 mL/min.The column was maintained at 30˚C and the detection was carried out at a wavelength of 261 nm (PDA, UV).The injection volume was 20 µL.

Stock and Working Standard Solutions
The stock solution of sitagliptin (5000 µg/mL) and metformin (50,000 µg/mL) was prepared by taking 50 mg and 500 mg of sitagliptin and metformin respectively into a 10 mL volumetric flask.Standard drugs were dissolved in 3 mL methanols and sonicated for 5 minutes followed by made up to the volume with the addition of methanol.From the stock solutions, working solutions of sitagliptin and metformin were prepared in 1 -1000 µg/mL and 10 -10,000 µg/mL concentration range, respectively.

Sample Preparation
Ten tablets were weighed and the coats were removed.An accurately weighed amount of finely powdered janumet tablets equivalent to 500 mg of metformin and 50 mg of sitagliptin were taken into a volumetric flask and 3 mL of methanol was added.Followed by sonication for 25 minutes and then made up to 10 mL with methanol.
The solutions were filtered and prepared dilutions to 1 µg/mL of sitagliptin and 10 µg/mL of metformin.

Method Validation
The method validation was performed in accordance with ICH guidelines [10].The parameters assessed were linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), precision, reproducibility and robustness.

Selectivity and Specificity
A representative chromatogram of the standard drugs is shown in Figure 1.No additional peaks were observed.Figure 2 shows the chromatograms of standard drug contain 98.15% and 99.4% of sitagliptin and metformin respectively.The retention time of metformin and sitagliptin were found at 4.9 and 6.1 min respectively.Assay (%) 98% -102% this is in accordance with ICH guidelines.Hence the developed method can be routinely employed for the simultaneous estimation of sitagliptin and metformin in the marketed formulations.Assay values of sitagliptin and metformin were presented in Table 1.

Linearity
Linear calibration curves with correlation coefficients greater than 0.9999 were obtained over a concentration range of 10 -50 µg/mL for sitagliptin and 20 -100 µg/mL for metformin.The typical equation of the calibration curves of the both drugs were obtained with r value 0.998.The results shown that within the concentration range indicated there was an excellent correlation between peak area ratio and each concentration of the sitagliptin and metformin.

Limit of Detection and Limit of Quantification
The limit of detection (LOD) and limit of quantification (LOQ) were calculated for sitagliptin and metformin according to ICH guidelines by taking standard deviation and slope from the calibration curve.LOD and LOQ were calculated by using below formulae LOD 3.3 SD slope; LOQ 10 SD slope − × − × LOD and LOQ values were presented in Table 2.

Precision
Precision of the analytical method was determined by replicating the process in triplicates at 3 different concentrations of sitagliptin and metformin (combination) and chromatograms were recorded.Precision was calculated as a percentage relative standard deviation.The relative standard deviation should be less than 2 according to ICH guidelines.The % relative standard deveiation of sitagliptin (2, 4, 6 µg/mL) and metfromin (20, 40, 60 µg/mL) was found less than 2%.Precision data of sitaglitin and metfromin were given in Table 3 and Table 4 respectively.

Accuracy and Recovery Studies
To the pre analyzed samples known amount of standard solutions in different concentration levels (50%, 100% and 150%) were added.For these solutions chromatograms were developed to record accuracy and recovery.The recoveries of sitagliptin and metformin were determined at 3 concentration levels (low, medium, high).Accuracy of the method was treated as percent recovery.Recoveries of sitagliptin (Table 5) and metformin (Table 6) were in between 98% -102%.This is in accordance with ICH guidelines.Therefore method was found to be accurate.

Conclusion
A simple, selective and sensitive reverse phase HPLC analysis method was developed for the simultaneous estimation of sitagliptin and metfromin in marketed formulation.The method was found economical and simple with involvement of few steps.The assay has been validated as per the ICH guideline and the results have shown that the method is sensitive, accurate and reproducible.Therefore, the developed method is found suitable for the simultaneous determination of sitagliptin and metformin in formulations.

Table 1 .
Assay of sitagliptin and metformin.

Table 4 .
Precision data of metformin.