Determination of metformin in human plasma using hydrophilic interaction liquid chromatography–tandem mass spectrometry

A sensitive, simple and rapid assay based on hydrophilic interaction liquid chromatography (HILIC) with tandem mass spectrometry was developed and validated for the quantitative analysis of metformin in human plasma using protein precipitation. Plasma samples were prepared using a protein precipitation solution containing acetonitrile, 0.5% formic acid and the internal standard, metformin-D6. The analytes were separated on a GL Sciences Inertsil HILIC column using an isocratic mobile phase consisting of water/acetonitrile (30:70, v/v) and 0.1% formic acid. Metformin and internal standard were recorded using multiple reaction monitoring in positive ion electrospray mode with transitions of m/z 130–71 and m/z 136–77, respectively. No endogenous components in plasma were found to interfere with metformin measurements. The lower limit of quantification (LLOQ) was 0.5 ng/mL (0.1 pg on-column). The linear range was 0.5–500 ng/mL with an average correlation coefficient of 0.999 using weighted (1/x2) linear least-squares regression. Dilutional linearity was evaluated up to 5000-fold dilution and the results indicate no influence on the accuracy of analysis. The absolute extraction recovery was 81% for metformin. Intra-day and inter-day precision (CV, %) ranged from 0.73% to 7.18%, and accuracy within ±10.98% from nominal. The analyte was found to be stable for at least 38 days at −20 and −80 °C, 24 h at room temperature, and stable for four freeze–thaw cycles. The processed extracts were stable for 88 h at 4 °C.