A rapid and sensitive UHPLC-MS/MS method for epalrestat detection in micro-volumes of human plasma for the first time

A rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for epalrestat detection in human plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used to quantify epalrestat and the internal standard epalrestat-d₅ in the negative ion mode using multiple reaction monitoring (MRM). After acetonitrile-mediated protein precipitation, chromatographic separation was achieved using a reversed-phase C₁₈ column (ACQUITY UPLC BEH, 2.1 × 50 mm, 1.7 μm; Waters Corp) with acetonitrile and 2 mM ammonium acetate in water as the mobile phase through gradient elution. The retention time of epalrestat was 0.92 min and the entire run time was only 2.00 min. The calibration curve was linear in the range 10.0–8.00 × 10³ ng/mL (<i>r</i> ≥ 0.99). The within-run and between-run relative standard deviations (RSDs) were &lt; 9.3%. The within-run and between-run relative errors (REs) ranged −8.4–4.2%. No significant matrix effects were observed and the recovery rate was high. The method was fully validated, including reinjection reproducibility in human plasma, and was successfully applied in a pharmacokinetic study, in which 100% incurred sample reanalysis met the criteria.