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.

Differences in early acetaminophen hepatotoxicity between obese ob/ob and db/db mice.

Clinical investigations suggest that hepatotoxicity after acetaminophen (APAP) overdose could be more severe in the context of obesity and nonalcoholic fatty liver disease. The pre-existence of fat accumulation and CYP2E1 induction could be major mechanisms accounting for such hepatic susceptibility. To explore this issue, experiments were performed in obese diabetic ob/ob and db/db mice. Preliminary investigations performed in male and female wild-type, ob/ob, and db/db mice showed a selective increase in hepatic CYP2E1 activity in female db/db mice. However, liver triglycerides in these animals were significantly lower compared with ob/ob mice. Next, APAP (500 mg/kg) was administered in female wild-type, ob/ob, and db/db mice, and investigations were carried out 0.5, 2, 4, and 8 h after APAP intoxication. Liver injury 8 h after APAP intoxication was higher in db/db mice, as assessed by plasma transaminases, liver histology, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. In db/db mice, however, the extent of hepatic glutathione depletion, levels of APAP-protein adducts, c-Jun N-terminal kinase activation, changes in gene expression, and mitochondrial DNA levels were not greater compared with the other genotypes. Furthermore, in the db/db genotype plasma lactate and ß-hydroxybutyrate were not specifically altered, whereas the plasma levels of APAP-glucuronide were intermediary between wild-type and ob/ob mice. Thus, early APAP-induced hepatotoxicity was greater in db/db than ob/ob mice, despite less severe fatty liver and similar basal levels of transaminases. Hepatic CYP2E1 induction could have an important pathogenic role when APAP-induced liver injury occurs in the context of obesity and related metabolic disorders.