title: Simple and rapid assay for acetaminophen and conjugated metabolites in low-volume serum samples.
abstract: The use of marker compounds for estimating drug metabolic capacity or pharmacokinetic parameters is common in the biological sciences. Often small laboratory animals are used and thus sample size is a limiting concern. In this report, we describe an assay we developed for measuring the concentration of acetaminophen and its conjugated metabolites in low-volume serum samples. Acetaminophen and metabolites were removed from 10 microl serum samples by a single-step 6% (v/v) perchloric acid deproteination using theophylline as internal standard. Samples were separated in a pH 2.2 sodium sulfate-acetonitrile mobile phase at a flow-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 microg/ml for acetaminophen and acetaminophen sulfate and 3.91-500 microg/ml for acetaminophen glucuronide. The assay was linear in the range of concentrations analyzed. 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 acetaminophen sulfate, respectively. Results from the experiments show that acetaminophen and its conjugated metabolites can easily and reproducibly be measured in low-volume serum samples and thus may offer an additional method to measure these compounds when the volume of biological samples may be limited.
authors.affiliation: Pharmaceutics Division, College of Pharmacy, The University of Texas at Austin, 78712-1074, USA. ljb@mail.utexas.edu
authors.forename: L J
authors.initials: LJ
authors.lastname: Brunner
authors.forename: S
authors.initials: S
authors.lastname: Bai
mesh_terms: D000082:Acetaminophen / Q000097:blood* / Q000378:metabolism; D018712:Analgesics, Non-Narcotic / Q000097:blood* / Q000378:metabolism; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008297:Male; D011786:Quality Control; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D018712:Analgesics, Non-Narcotic; D000082:Acetaminophen