Reversed-phase chromatography of urinary metabolites of paracetamol using ion suppression and ion pairing

High-performance liquid chromatography (HPLC) has proven particularly useful for the study of paracetamol metabolism. Two alternative methods were developed using reversed-phase C₁₈ columns. A rapid ion suppression technique was used for the analysis of free paracetamol, paracetamol mercapturic acid and cysteine conjugate in urine samples obtained from isolated perfused rat kidney preparations, which has conveniently demonstrated the oxidative metabolic capacity of the kidney towards paracetamol. A somewhat longer, but higher resolution, ion-pair HPLC procedure was developed for the analysis of paracetamol metabolites in urine samples from experimental animals. The ion-pairing solvent was composed of tetrabutylammonium hydroxide, Tris and EDTA buffered to pH 7.2 with phosphoric acid. Gradient programming was further used to enhance resolution. Using this system two new metabolites, the sulphate and glucuronide conjugates of 3-thiomethyl-paracetamol were detected and routinely determined along with other known paracetamol metabolites, viz. free paracetamol, paracetamol sulphate, glucuronide, mercapturic acid, and cysteine conjugates, 3-methoxyparacetamol glucuronide and sulphate, p-aminophenol and its O-glucuronide and O-sulphate conjugates. Phenolic O-substituted glucuronide and sulphate conjugates of N-hydroxyparacetamol were also separated.