Bioanalytical Method Development and Validation for the Estimation of Metformin and Vildagliptin in K3EDTA Human...

A unique liquid chromatography-tendon mass spectrometric technique for the determination of metformin and vildagliptin in K3EDTA human plasma was developed and verified as per the USFDA guidelines of bioanalysis.

The chromatographic separation was achieved using a Cosmosil CN (150 x 4.6 mm, 5 μm) column with an isocratic elution pattern using 10 mM ammonium formate (pH 5.0) and methanol in the ratio of 30:70 v/v as a mobile phase. A mass spectrometer coupled with an electrospray ionization (ESI) source operating in the positive ion was used for detection. Data were obtained in the multi-reaction monitoring (MRM) acquisition mode. Metformin D6 and vildagliptin D7 were used as internal standards, with the flow rate at 1.0 mL/min throughout the experiment. The drugs were extracted by solid phase extraction (SPE) packed with Phenomenex Strata-X. Extraction of the drug was achieved using methanol: 5 mM sodium lauryl sulphate solvent mixture in equal proportions.

The retention time for MET and VLG were 3.2 and 3.8 minutes individually. The drugs were extracted by SPE with good recovery of 89.44% and 87.57% for metformin and ISTD and 92.26% and 89.58% for vildagliptin and ISTD, respectively. Sample elution was performed using solid phase extraction (SPE), and this technique produced very pure extracts with good recovery rates. A liner calibration curve was found in the range of 0.5-400 ng/mL for MET and 0.2-160 ng/mL for VLG with a correlation coefficient r2 > 0.99.

The aforementioned technique is reliable and effective for monitoring bioequivalence investigations in human participants.