LC-MS/MS for measuring metformin concentration in plasma

Metformin has high apparent volume of distribution and the protein binding is negligible. Therefore, simple protein precipitation was utilized for plasma metformin extraction. Pooled mice plasma (CD-1 (ICR) MSE00PLK2, BioIVT) was selected as blank matrix for standards and quality control samples. Sample preparation procedure was adopted from literatures with minor modifications. In brief, to 20 μL of blank pooled plasma or mouse plasma samples, 20 μL of 50% methanol-water or standard solutions were added followed by addition of 360 μL of ice-cold acetonitrile solution containing IS (111 nM metformin-D6). The samples mix were vortexed for 2 min then sonicated for 1 min and centrifuged at 6100 g for 10 min. Supernatants were transferred into a new set 96- well plate, and 10 μL were injected for LC-MS/MS analysis. A 2 μg/mL metformin stock solution was prepared in 50% methanol-water. The solution was then serially diluted with 50% methanol-water to obtain standard working solutions over a concentration range of 10–2000 ng/mL. The quality control (QC) samples were prepared in three concentrations: 10, 100, and 500 ng/mL.

LC-MS/MS system was performed using an Agilent 1290 Infinity HPLC and HiP ALS (Autosampler) coupled with AB Sciex 6500 Q-Trap mass spectrometer. All data were acquired using Analyst (AB SCIEX v1.7.1). Metformin from mice plasma samples were separated on a Waters XBridge Amide column (2.1 × 150 mm, 3.5 μm) at 40°C. The mobile phase A was 20 mM ammonium acetate buffer in water and 100% acetonitrile as mobile phase B. A total 7 min gradient run with a flow rate of 0.8 mL/min was implemented. The chromatographic runs started with 95% mobile phase B for 2 min, then gradually increased mobile phase A from 5% to 95% in 1.5 min, kept the phase A at 95% for 1 min and reduced to 5% in 1.5 min, finally equilibrate the column at 95% phase B for 2 min. The mass spectrometer was operated in a positive mode using scheduled multiple reaction morning (sMRM). The ion source parameters were as follow: curtain gas (CUR) at 37, collision gas (CAD) at high, ionspray voltage (IS) at 5 kV, temperature (TEM) at 500, ion source gas 1 (GS1) at 60, ion source gas 2 (GS2) at 50, MRM detection window at 120 s, target scan time (per sMRM expt) at 0.15 s, the retention time (RT) for both metformin and metformin-D6 were at 3.26 min, Q1 were 130.2 and 136.2 for metformin and metformin-D6 respectively, Q3 was 60.2, declustering potential (DP) at 100, collision energy (CE) at 13, entrance potential (EP) at 10, and collision cell exit potential (CXP) at 17.