Determination of paracetamol and its metabolites in urine by high-performance liquid chromatography using ion-pair...

Paracetamol (P) and its four main metabolites, the sulphate (S), glucuronide (G), cysteine (C) and mercapturic acid (M) conjugates, are separated on ODS/TMS silica using a standard eluent, water-methanol-formic acid (86:14:0.1, v/v/v), in the order S, G, P, C, M. On addition of the ion-paring cations dioctylammonium (DOA) and tetrabutylammonium (TBA), the retention of S is vastly increased while those of G and M are substantially increased and that is C is reduced. The excessive retention of S and to a lesser extent that of M and G can be controlled by the addition of a suitable concentration of a co-ion such as nitrate. The order of elution is then G, C, P, M, S, although this can be varied by adjusting the amount of nitrate present. Loading of DOA is slow as it is very strongly adsorbed and typical concentrations in the eluent are below 7 mg/l. Loading by TBA is rapid with typical concentrations being around 200 mg/l. The effects of added co-ions such as nitrate can be explained in terms of simple ion-pair equilibria. Equilibration with respect to added salts is rapid. Application of the technique to analysis of therapeutic and overdose urines shows the presence of at least three additional metabolites, one of which is identified by mass spectrometry as most probably 3-methoxyparacetamol. Another appears to be a methoxymercapturic acid derivative. There is further evidence for a group of metabolites that elute unresolved from overdose urines as a broad band after the main metabolites.