A rapid and efficient approach for quantifying metformin in human plasma using Ultra-High performance liquid chromatography with UV-Detection: Relevance in Non-Diabetes clinical studies

Metformin, a first-line drug to treat type-2 diabetes, has gained attention in various non-diabetes studies, such as anti-aging, reduced cancer incidence and mortality, and reduced symptomatic viral infection due to its immunomodulatory properties. This study aims to develop and validate a simple and cost-effective ultra-high-performance liquid chromatography method with UV detection (uHPLC-UV) for quantifying metformin in human plasma amidst operational challenges and restricted accessibility to mass spectrometry. Plasma samples were protein precipitated with acetonitrile, and metformin was separated from endogenous plasma components using a Waters Acquity UPLC BEH Amide column, 1.8 µm, (2.1 x 100 mm) with gradient elution using 0.1 M potassium phosphate adjusted with phosphoric acid to pH 5.8, and acetonitrile as mobile phases and detected at 240 nm. Method validation was performed according to U.S. FDA guidelines. Total run time achieved was 3 min and linearity was demonstrated over the range of 0.1–40 mg/L based on linear regression using 1/x weighting. The lowest quantitation limit of 0.1 mg/L was achieved. Excellent recoveries (90.3–98.4 %) and negligible matrix factors (94.6–108.1 %) were observed in the sample preparation, negating the need for using any internal standard. Finally, the developed assay was utilised to measure metformin plasma levels from healthy volunteers recruited in a randomised, double-blind, placebo-controlled study investigating potential immunomodulatory effects of metformin influence on yellow fever viremia.