Quantitative Analysis of Acetaminophen and its Primary Metabolites in Small Plasma Volumes by Liquid Chromatography-Tandem Mass Spectrometry

A fast and sensitive liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was developed and validated for the simultaneous determination of acetaminophen (APAP) and its glucuronide and sulfate metabolites (APAP-GLU and APAP-SUL) in small plasma volumes. This method included a simple step of sample preparation and a chromatographic separation on an LC–MS-MS system equipped with an electrospray ionization source and a tandem triple quadrupole mass spectrometer in multiple reaction monitoring mode. The analytes and internal standard, APAP deuterated analog, were separated on a C18 column (3.0 µm, 2.1 × 100 mm), using aqueous 1% formic acid and methanol (80:20, v/v) as the mobile phase. The LC–MS-MS method was validated for accuracy, precision, linearity, extraction efficiency, process efficiency and matrix effect. Calibration curves were obtained by fortifying drug-free plasma and ranges of linearity were set between 0.25–20 mg/L. The mean correlation coefficients, r2, were >0.99 for APAP and its metabolites. The inter-day and intra-day precision values were less than 11.75 and 13.03%, respectively, at the lower limit of quantification concentration. The usability of the method was demonstrated by studying APAP metabolism in C57BL/6J wild-type and obese ob/ob female mice, in which only small plasma volumes were available. The results showed that APAP glucuronidation was enhanced in obese mice, suggesting that changes in APAP metabolism could modify its toxicity in obesity and related fatty liver disease.