Determination of bradykinin in rat urine by coupled-column high pressure liquid chromatography with precolumn derivatization with a water-soluble fluorogenic reagent

Bradykinin (BK) in rat urine was determined by coupled-column HPLC with precolumn fluorogenic derivatization with a water-soluble reagent, 3-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)benzenesulfonic acid (m-BS-ABD-F). The derivatization of BK with m-BS-ABD-F was completed at 70 degreesC for 100 min and gave only a single peak of BK derivative in addition to the peaks of the blank. The hydrophilicity of the derivatization reagent effectively prevented the adsorption of BK during the sample pretreatment and improved the recovery of BK. Good linearity was shown between the amount of BK spiked in urine (0-10 pmol) and the peak area of the BK derivatives (correlation coefficients >0.999), and the detection limits of the BK derivative were 35 fmol (S/N = 3). The precisions (cv, %) of intra- and interday assay were not more than 5.5% and the accuracies were in the range of 95.3-111% (1 and 5 pmol of BK in urine, n = 3). Although the peak regarded as that of the BK derivative rapidly decreased after incubation at 37 degreesC, addition of urinary kininase inhibitors to the urine samples drastically suppressed the decrease of this peak, confirming that the identified peak was that of the BK derivative. The urinary kinin excretion in male SD rats (9-11 weeks old) determined by the present method was 56.0 +/- 22.1 pg(min/kg (mean +/- SE, n = 5). (C) 2001 Academic Press.