Urinary mesna and total mesna measurement by high performance liquid chromatography with ultraviolet detection

We describe in this report a method for determination of mesna and total mesna in urine by high performance liquid chromatography with ultraviolet detection. The method involves a treatment of the urine sample with tri-n-butylphosphine in order to convert mesna disulfides to its reduced counterpart mesna, ultraviolet labelling with 2-chloro-1-methylquinoluinium tetrafluoroborate, reversed-phase HPLC separation, and detection and quantitation at 350nm. The result corresponds to total mesna that is sum of mesna, dimesna and its mixed disulfides with endogenous thiols. For determination of mesna the reduction step is omitted. Content of disulfide forms of mesna can be calculated by subtracting the concentration of mesna from the total mesna concentration. The separation of 2-S-quinolinium derivatives of mesna from those of endogenous urinary thiols and internal standard was achieved on an analytical Waters Nova-Pak C18 (150mmx3.9mm, 5mum) column. A mixture of an aqueous solution of pH 2.3, 0.05M trichloroacetic acid and acetonitrile (88:12, v/v) was used as a mobile phase at flow rate of 1.2ml/min and ambient temperature. The assay for mesna and total mesna in urine was proved to be linear over the studied ranges of 0.2-30 and 0.2-800nmol/ml urine, respectively. The mean recoveries over the calibration ranges were 95.4% for mesna and 99.7% for total mesna. The lower limits of detection and quantitation were 0.1 and 0.2nmol/ml for both the procedures, respectively. The imprecision did not exceed 8.5%. No interference from endogenous substances was observed.