Development of a UPLC-MS/MS method for quantitation of metronidazole and 2-hydroxy metronidazole in human plasma and its application to a pharmacokinetic study

Abstract An ultra-performance liquid-chromatography mass-spectrometry (UPLC-MS/MS) method for simultaneous quantitation of metronidazole and 2-hydroxymetronidazole in human plasma was developed and validated. Metronidazole and 2-hydroxymetronidazole were extracted from a small volume of human plasma (10 μL) by hydrophilic lipophilic balanced solid phase extraction on 96-well μ-elution plates. Chromatographic separation of analytes was achieved on an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) using gradient elution with a blend of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.25 mL/min. Mass spectrometric detection was achieved using multiple reaction monitoring (MRM) in positive-ion electrospray-ionization (ESI) mode. Ion transitions were optimized at m / z 171.85->127.9 for metronidazole and m / z 187.9->125.9 for 2-hydroxymetronidazole. The assay was linear for both analytes over the concentration range of 0.1–300 μM; intra- and inter-assay precisions and accuracies were