Automated liquid chromatographic method for the determination of paracetamol and six metabolites in human urine.

An automated liquid chromatographic method, with a coefficient of variation for total imprecision of less than 4%, has been developed for the quantitative determination of paracetamol, paracetamol-4-glucuronide, paracetamol-4-sulphate, paracetamol-3-cysteine, paracetamol-3-mercapturate, 3-hydroxyparacetamol-3-sulphate and 3-methoxyparacetamol-4-sulphate in urine samples. The gradient elution system was based on 0.067 M phosphate buffer (pH 2.0) and acetonitrile on an octadecylsilica column. The on-column detection limit using an ultraviolet detector at 254 nm for each of the compounds using 3-hydroxyacetanilide as internal standard was of the order of 10-50 ng from urine and 2-10 ng from water. Application of the method to 24-h urine samples from subjects who had received a therapeutic dose of the drug confirmed the findings of previous studies for the importance of the glucuronide, sulphate, mercapturate and cysteine conjugates. 3-Hydroxyparacetamol-3-sulphate was shown to be present in the urine of all volunteers and to account for up to 5% of the dose. 3-Methoxyparacetamol-4-sulphate was not detected in any urine samples and if present as a metabolite must account for less than 0.1% of the dose.