Determination of salicylamide and five metabolites in biological fluids by high‐performance liquid chromatography

Two high‐performance liquid chromatographic (HPLC) assay procedures were developed for the determination of salicylamide and its metabolites in serum, urine, and saliva. One method involves reverse‐phase ion‐pair chromatography and UV detection, and is used to determine salicylamide, salicylamide glucuronide, and salicylamide sulfate. The other method, with a different mobile phase and without the ion‐pairing reagent, is used to determine gentisamide (the hydroxylated metabolite of salicylamide), gentisamide glucuronide, and gentisamide sulfate. The assays are performed by direct injection of the sample after protein precipitation with ethanol containing the internal standard. Increased sensitivity for the determination of low concentrations of salicylamide is obtained by organic extraction of this drug from serum or saliva. Calibration curves for the conjugates of salicylamide and gentisamide were obtained, in the absence of authentic standards, by partial enzymatic hydrolysis, using the decrease of the conjugate peaks and the concomitant increase of free salicylamide or gentisamide concentrations to determine peak area ratio–concentration relationships. Application of the HPLC assay procedures to the determination of salicylamide excretion products in the urine of three normal human subjects resulted in 98.6% (range: 97.1–100.1%) recovery of a 1‐g oral dose of the drug. All five metabolites of salicylamide were found in urine, but only salicylamide glucuronide, salicylamide sulfate, and gentisamide glucuronide were found consistently and in appreciable quantities. Salicylamide and all of its metabolites except gentisamide sulfate were found in human and rat serum, and unconjugated salicylamide as well as gentisamide were found in human saliva.