The estimation of paracetamol and its major metabolites in both plasma and urine by a single high-performance liquid chromatography assay.

Many analytical methods exist for the assay of paracetamol in biological fluids, including colorimetry with chemical derivatization, direct spectrophotometry, chromatographic methods and immunoassays. Their development has been largely driven by the needs of clinical toxicology requiring the rapid, reliable and highly specific estimation of paracetamol in plasma samples to determine the need for antidote therapy. However, for in vivo metabolism studies, a specific assay method which can provide measurements of paracetamol and its metabolites in both plasma and urine is desired. A reversed-phase HPLC method with UV detection at 254 nm was developed to fulfil these requirements. The assay involves minimum sample preparation with a relatively short run time. The solvent system involves a simple isocratic elution with a composition of 0.1 M potassium dihydrogen orthophosphate-acetic acid-propan-2-ol, (100:0.1:0.75, v/v/v). The reproducibility of the assay was high with an inter-assay RSD of 0.2-1.7% for urinary paracetamol concentrations of 5-500 micrograms ml-1 and 0.1-3.3% for plasma concentrations between 5 and 25 micrograms ml-1. A similarly high degree of precision was found for the glucuronide, sulphate, cysteine and mercapturate metabolites of paracetamol. The same assay can be used to analyse both plasma and urine samples and thus was employed for studies on the metabolism of paracetamol in healthy subjects and in patients with various diseases.