A Validated High-Performance Liquid Chromatographic Method for the Determination Of Paracetamol and Its Major Metabolites in Urine

Abstract

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method employing UV-detection (248 nm), 5 μm Resolve C18 as stationary phase (in a 8 × 100 mm Waters Radial-Pak cartridge) and 0.1 M potassium phosphate monobasic/methanol/glacial acetic acid (95:4:1, v/v/v) as mobile phase was developed for the rapid and simultaneous determination of paracetamol and its major metabolites (its glucuronide and sulphate conjugates) in urine. The method requires only minimal sample preparation and chromatographic run time is only 6 min. For determination of paracetamol, the precision (inter-assay RSD ranged from 1.60–0.66% between 5 and 100 μg mL⁻¹) and limits of detection (2 ng mL⁻¹) were satisfactory, as were these parameters for determination of the major metabolites. In the course of studies on paracetamol bioavailability and metabolism, over 1500 samples have been assayed using this method. Herein we report its use for monitoring of the levels of paracetamol and its major metabolites in the urine of fifteen normal healthy volunteers given a single oral dose of 500 mg.