Bioanalytical Method Using Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Mass...

Metformin is the first-line medicine for the treatment of type 2 diabetes. Drug interactions between metformin and other drugs, food, or beverages cannot only cause changes in the pharmacokinetic profiles but also affect the efficacy of metformin. The purpose of this study was to develop a rapid and reliable bioanalytical method for the detection of plasma metformin concentration in humans. To remove interfering substances in plasma, acidified acetonitrile (acetonitrile containing 0.1% formic acid) was added to samples. Ultra-high-performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (HRMS) was used to analyze metformin and its internal standard (metformin-d6). Analyte separation was performed on a BEH HILIC analytical column (100 × 2.1 mm, 1.7 μm) using a gradient elution of 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The total chromatographic run time was 2 min. The developed method was validated for its linearity, accuracy and precision, selectivity (signal of interfering substance; analyte, lower limit of quantification (LLOQ) ≤ 20%; IS, IS ≤ 5%), sensitivity (LLOQ, 5 ng/mL; S/N ratio ≥ 10), stability (low quality control (LQC, 15 ng/mL), 2.95-14.19%; high quality control (HQC, 1600 ng/mL), -9.49-15.10%), dilution integrity (diluted QC (4000 ng/mL); 10-folds diluted QC (400 ng/mL); 5-folds diluted QC (800 ng/mL); accuracy, 81.30-91.98%; precision, ≤4.47%), carry-over (signal of double blank; analyte, LLOQ ≤20%; IS, IS ≤5%), and matrix effect (LQC, 10.109%; HQC, 12.271%) under various conditions. The constructed calibration curves were shown linear in the concentration range of 5-2000 ng/mL, with within- and between-run precision values of <8.19% and accuracy in the range of 91.13-105.25%. The plasma metformin concentration of 16 healthy subjects was successfully measured by applying the validated bioanalytical method.