Quantification of metronidazole in healthy subjects' feces using an LC-MS/MS method

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC MS/MS) method was developed for the quantification of metronidazole (MTZ) in human feces. The analyte was recovered from feces after liquid-liquid extraction with ethyl acetate and separated on Waters Symmetry® C₁₈ (100 × 4.6 mm, 5μm) column using 0.1% formic acid in water and acetonitrile (40:60, v/v) as the mobile phase. A stable-deuterated internal standard metronidazole-d4 (MTZ-d4) was used in the study. Mass analysis was performed on a triple quadrupole mass spectrometer in the positive electrospray ionization mode. A linear response function of MTZ was established in the concentration range of 0.50-250 ng/g, based on dry mass. The mean extraction recovery of MTZ (97.28%) and MTZ-d4 (96.76%) from spiked feces samples was consistent at higher as well as lower concentrations. Post-column infusion analysis showed no ion-suppression/enhancement effects and the mean IS-normalized matrix factor ranged from 0.986 to 1.013. Spiked feces samples stored at -20 and - 70°C for long-term stability were stable for at least 3 months, while extracted samples (dry and wet extracts) were stable up to 24 h. The method was applied to determine MTZ in feces of 12 healthy Indian subjects.