LC-MS/MS quantitation of metformin, phenformin, and galegine.

Plasma or frozen tissue was added to 0.1 mL of water with 0.1% formic acid and 0.15 mL of methanol containing 0.4 μmol of internal standard (D₆-metformin for metformin and galegine and D₅-phenformin for phenformin). Plasma samples were vortexed, and tissue samples were homogenized followed by centrifugation. The supernatant was dried under a stream of nitrogen gas, resuspended with 0.3 mL of water with 0.1% formic acid, and filtered with a nanosep 100K centrifugal device before LC-MS/MS analysis.

An ESI source with a positive mode was used for the analysis of metformin, phenformin, and galegine, and the ion-pair-dependent parameters were optimized. The optimized parameters are: curtain gas (14 V), collision gas (medium), ionization potential (5,000 V), probe temperature (450 °C), ion source gas 1 (60 V), ion source gas 2 (60 V), and EP (10 V). The other compound-dependent parameters were also summarized in SI Appendix, Table S1 for phenformin, galegine, and D₆-metformin. A C₈ reverse-phase LiChrosorb HPLC column (100 mm × 3 mm) was used with a mixed solvent of 10 mM ammonium acetate aqueous solution and pure acetonitrile. Concentrations were calculated from the peak area ratios of metformin, phenformin, and galegine vs. their internal standard, according to the corresponding calibration curves.