The quantification of paracetamol, paracetamol glucuronide and paracetamol sulphate in plasma and urine using a single high-performance liquid chromatography assay

A range of analytical methods exist for the determination of paracetamol in biological fluids. However, to understand the fate of paracetamol and the effect of other drugs on its disposition in vivo, the major metabolites require quantification in urine and plasma. A method to simultaneously quantify paracetamol, paracetamol glucuronide (PG) and paracetamol sulphate (PS) in plasma and urine with superior sensitivity is therefore desired, especially if the volume of plasma available is low. A simple isocratic reverse phase high-performance liquid chromatography (HPLC) assay with spectrophotometric detection has been developed. The method, requiring only 100 μl of plasma and 50 μl of urine, utilizes a reversed-phase C18 column, a wavelength of 254 nm for detection and a mobile phase composed of potassium dihydrogen orthophosphate (0.1 M)–isopropanol–tetrahydrofuran (THF) (100:1.5:0.1, v/v/v) adjusted to pH 3.7 with phosphoric acid. The method is sensitive and linear in plasma within a concentration range from 0.4 to 200 μM for paracetamol, PG and PS. For PG and PS in urine, the method is sensitive and linear within a concentration range from 100 to 20,000 μM. Over these ranges, accuracy and precision were less than 12%. The assay has been used to measure concentrations of paracetamol and the two metabolites in plasma collected by finger-prick sampling and of the metabolites in urine from healthy volunteers administered a single oral dose of 1000 mg of paracetamol.