Fast liquid chromatographic-tandem mass spectrometric (LC-MS-MS) determination of metformin in plasma samples

Liquid chromatographic-tandem mass spectrometric (LC-MS-MS) methods for the determination of metformin in plasma from different species are presented. The first method employed a YMC cyano 2mm x 50 mm, 3 microm analytical column. For minimum sample preparation direct injection of samples after protein precipitation was performed. The polar column used with highly organic mobile phases provided a normal phase retention mechanism. The elution conditions were optimized to obtain reproducible peak areas and good peak shape. A step gradient from 100% acetonitrile to acetonitrile-water 80:20 (v/v) containing 10mM ammonium acetate and 1% acetic acid was applied, leading to a sample-to-sample cycle time of 2 min. In a second method, a column-switching LC-MS-MS assay for on-line trapping was developed. The analyte and internal standard were trapped on a YMC cyano 2 mm x 10 mm, 5 microm column using acetonitrile-methanol 95:5 (v/v). Elution was performed isocratically in back-flush mode on to the analytical column (YMC cyano 2 mm x 50 mm, 3 microm) using 10 mM ammonium acetate in acetonitrile-water 80:20 (v/v) with 1% formic acid. With this approach, the signal-to-noise ratio was improved and the run time could be shortened to 1 min. Calibration samples were prepared in the matrix to be assayed in the range of 10-10,000 ng/ml. Quality control (QC) samples were prepared at 40, 400 and 4000 ng/ml and interspersed with the unknown study samples in the assays. Deviations for precision and accuracy were less than 20% for the lower limit of quantification (LLOQ) and low QC sample and less than 15% for other calibrators and QCs.