Bioanalytical Method Development and Validation for the Estimation of Metformin and Vildagliptin in K3EDTA Human Plasma Using HPLC-ESI-MS/MS
Binit Patel, Shalin Parikh, Ravi Patel, Khushbu Patel, Pinak Patel, Pravinkumar Darji, Archita Patel, Samir PatelObjective:
A unique liquid chromatography-tendon mass spectrometric tech-nique for the determination of metformin and vildagliptin in K3EDTA human plasma was developed and verified as per the USFDA guidelines of bioanalysis.
Methods:
The chromatographic separation was achieved using a Cosmosil CN (150 x 4.6mm, 5μm) 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) acquisi-tion 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 propor-tions.
Results:
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 met-formin 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 corre-lation coefficient r2 > 0.99.
Conclusion:
The aforementioned technique is reliable and effective for monitoring bio-equivalence investigations in human participants.