Complete separation of urinary metabolites of paracetamol and substituted paracetamols by reversed-phase ion-pair high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic procedure has been developed for the separation of thirteen urinary metabolites of the analgesic drug paracetamol. The method involved the use of radially compressed columns packed with octadecylsilica with a particle diameter of 5 micron. Metabolites were chromatographed by linear gradient elution using an ion-pair solvent system composed of tetrabutylammonium hydroxide and Tris buffered to pH 5.0 with phosphoric acid, and acetonitrile as the organic solvent. Analyses can be performed at the rate of three per hour. This method enables the direct identification of sulphate and glucuronide conjugates of 3-thiomethylparacetamol and 3-thiomethylparacetamol sulphoxide which have only previously been detected following enzyme hydrolysis of urine samples. The application of this fully optimised separation to the study of the metabolism of substituted paracetamols is also discussed.