Simple and rapid assay for acetaminophen and conjugated metabolites in low-volume serum samples

The use of marker compounds for estimating drug metabolic capacity or pharm acokinetic parameters is common in the biological sciences. Often small lab oratory animals are used and thus sample size is a Limiting concern. In thi s report, we describe an assay we developed for measuring the concentration of acetaminophen and its conjugated metabolites in low-volume serum sample s. Acetaminophen and metabolites were removed from 10 mu l serum samples by a single-step 6% (v/v) perchloric acid deproteination using theophylline a s internal standard. Samples were separated in a pH 2.2 sodium sulfate-acet onitrile mobile phase at a how-rate of 1.5 ml/min on a 15 cm octadecylsilyl column at room temperature. Analytes were detected at a wavelength of 254 nm. The resulting chromatograms showed no interfering peaks from endogenous serum components. The concentration ranges measured were 1.56-200 mu g/ml for acetaminophen and acetaminophen sulfate and 3.91-500 mu g/ml for acetam inophen glucuronide. The assay was linear in the range of concentrations an alyzed. The intra-day and inter-day coefficient of variation ranged from 0. 4 to 8.2% and 0.2 to 12.3% for acetaminophen, 0.5 to 12.9% and 0.3 to 16.1% for acetaminophen glucuronide, and 0.4 to 8.1% and 0.2 to 14.3% for acetam inophen sulfate, respectively. Results from the experiments show that aceta minophen and its conjugated metabolites can easily and reproducibly be meas ured in low-volume serum samples and thus may offer an additional method to measure these compounds when the volume of biological samples may be limit ed. (C) 1999 Elsevier Science B.V. All rights reserved.