Pentafluorophenylprophyl Ligand-based Liquid Chromatography-Tandem Mass Spectrometric Method for Rapid and Reproducible Determination of Metformin in Human Plasma

Abstract

This paper describes first development and validation of pentafluorophenylprophyl ligand-based liquid chromatography coupled to tandem mass spectrometry (PFPLC-MS/MS) method to determine metformin, a highly polar compound, in human plasma. Metformin and Phenformin (internal standard) were extracted from human plasma 50 <TEX>${\mu}L$</TEX> with a single-step protein precipitation. The chromatographic separation was performed using a linear gradient elution of mobile phase involving 5.0 mM ammonium formate solution with 0.1% formic acid (A) and acetonitrile (B) over 3.0 min of run time on a Phenomenex Luna PFP column. The detection was performed using a triple-quadrupole tandem mass spectrometer (Waters Quattro micro) with electrospray ionization in the mode of positive ionization and multiple-reaction monitoring (MRM). The developed method was validated with 5.0 ng/mL of lower limit of quantification (LLOQ). The calibration curve was linear over 5-3000 ng/mL of the concentration range (<TEX>$R^2$</TEX> > 0.99). The specificity, selectivity, carry-over effect, precision, accuracy and stability of the method met the acceptance criteria. The method developed in this study had had rapidness, simplicity and ruggedness. The reliable method was successfully applied to high throughput analysis of real samples for a practical purpose of a pharmacokinetic study.