Improved high-performance liquid chromatographic separation of urinary paracetamol metabolites using radially...

Methods have been adapted for the high-performance liquid chromatographic (HPLC) analysis of urinary paracetamol metabolites on radial compression columns. Enhanced resolution and decreased analysis time were two major advances. Various modifications to existing methods were made to counter the effect of the different C18 surface. Thus in ion suppression HPLC the addition of triethylamine at pH 3.0 (phosphate buffer) was necessary to block residual hydroxyl sites, while in ion-pair HPLC a higher tetrabutyl-ammonium hydroxide concentration of 0.01 M at pH 5.0 was used to enhance selectivity. The methods were successfully applied to the study of the metabolism of paracetamol, its glutathione conjugate and 3-thiomethylparacetamol in Sprague-Dawley rats. 3-Thiomethyl-paracetamol sulphoxide and its glucuronide and sulphate conjugates were shown to be metabolites of both 3-thiomethylparacetamol and paracetamol. 3-Thiomethylparacetamol sulphate was unresolved from the sulphates of paracetamol and 3-methoxyparacetamol in ion-pair HPLC. This raises a previously unrecognised problem in which the peak normally attributed to paracetamol sulphate contains metabolites arising from an oxidative metabolic pathway. Elevated levels of 3-methoxyparacetamol conjugates were found in human overdose urine and to some extent in analgesic nephropathy.