Determination of acetaminophen and its main metabolites in urine by capillary electrophoresis hyphenated to mass spectrometry

Abstract In this study, a capillary electrophoresis-tandem mass spectrometry method combining efficient separation and sensitive detection has been developed and validated, for the first time, to quantify acetaminophen and five of its metabolites in urine samples. Optimization of the method has led us to perform detection in positive ESI mode using MeOH-ammonium hydroxide (0.1%) (50:50, v/v) as sheath liquid. Moreover, optimal separation has been obtained in less than 9 min after anodic injection, using an ammonium acetate solution (40 mM, pH 10) as BGE. It was shown that the dilution solvent and the dilution factor to use for sample preparation are critical parameters to avoid peak splitting, to gain in sensitivity and then to obtain an effective analysis method. While a 200-fold factor dilution was shown to be suitable for quantitation of acetaminophen, acetaminophen mercapturate, acetaminophen sulfate and acetaminophen glucuronide, a 20-fold dilution was finally selected for methoxy-acetaminophen and 3-methylthioacetaminophen analysis, thus requiring two successive analyses to be carried out in order to quantify all metabolites. Hyphenation of CE with MS/MS versus UV permits to improve LOQ (10–20-fold factor with respect to previous works for acetaminophen, acetaminophen sulfate and acetaminophen glucuronide). Moreover, use of CE versus HPLC, permits to quantify two additional metabolites, i.e. 3-methylthio-acetaminophen and methoxy-acetaminophen. The method has been validated using the accuracy profile approach with a total error (accuracy) included in the ± 20% range. Thereby, the method allows the quantitation of acetaminophen and acetaminophen mercapturate in the range (0.1–1 mg mL-1), and of acetaminophen sulfate, methoxy-acetaminophen, acetaminophen glutathione and 3-methylthio-acetaminophen in the ranges (0.5–5 mg mL-1), (0.025–0.4 mg mL-1), (9.22–30 mg mL-1) and (0.073–0.4 mg mL-1), respectively. The method was finally applied to the analysis of urine samples of eighteen patients belonging to three different inclusion groups of the ongoing clinical trial, demonstrating that the method is suitable to highlight different metabolic profiles. This work will be subsequently extended to the analysis two hundred and seventy urine samples from patients included in a clinical trial dedicated to the study of acetaminophen metabolism changes after hepatic resection.