Measurement of reduced and total mercaptamine in urine using liquid chromatography with ultraviolet detection.

A simple liquid chromatographic method for the determination of reduced and total mercaptamine in human urine is described. The method is based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet-absorbance detection at 355 nm. Total mercaptamine was determined by reductive conversion of its oxidized fraction to the thiol form before the derivatization step. Baseline separation was achieved on an analytical Zorbax SB C(18) (5 microm, 150 x 4.6 mm) column with a mobile phase consisting of pH 2.0 0.05 mol L(-1) trichloroacetic acid buffer (component A) and acetonitrile (component B) pumped at 1.2 mL min(-1). Gradient elution was used: 0-3 min 12% B, 3-9 min 12-30% B, 9-12 min 30-12% B. The response of the detector was linear within the ranges studied, from 0.1 to 50 micromol L(-1) for reduced mercaptamine and from 0.4 to 400 micromol L(-1) for total mercaptamine. The imprecision ranges for reduced and total mercaptamine were within 1.45-11.71 and 0.73-10.61%, respectively. The analytical accuracy for determined compounds was from 98.79 to 109.77%. The lower limits of detection and quantitation were 0.05 and 0.1 micromol L(-1) of urine for reduced mercaptamine, and 0.2 and 0.4 micromol L(-1) of urine for total mercaptamine, respectively. This method can be used for routine clinical monitoring of the title thiol-drug and its reduced and oxidized fractions. Moreover, cysteine and cysteinylglycine can be measured concurrently, if needed.