Quantification of BK1-5, the stable bradykinin plasma metabolite in humans, by a highly accurate liquid-chromatographic tandem mass spectrometric assay

Bradykinin is a vasoactive nonapeptide involved in cardiorenal physiology a nd inflammatory states. It has been linked to the pathophysiology of hypert ension and diabetes. Correlating levels of bradykinin with disease states h as been hampered by its rapid degradation, artifactual production during bl ood sampling, and nonspecific radioimmunoassay techniques. We previously id entified BK1-5 as the stable in vivo plasma metabolite of systemic bradykin in in humans. We now report a sensitive and specific assay method for BK1-5 in human blood utilizing liquid chromatography-tandem mass spectrometry (M S) with electrospray ionization. [C-13(2),N-15]Glycine was incorporated int o chemically synthesized BK1-5 for use as an internal standard. Blood sampl es (5 mi were collected into 15-ml chilled ethanol to prevent artifactual k inin production and degradation. BK1-5 in ethanolic plasma supernatant was purified on a polymeric solid phase extraction cartridge. MS analysis was i n the selective reaction monitoring mode, Precision of the assay is +/-7.5% and accuracy is 99%. Recovery of BK1-5 through sample preparation was 43% and the lower limit of detection is 4 fmol/ml blood, Concentrations of BK1- 5 in 12 normal volunteers were 44.2 +/- 7.1 fmol/ml blood (mean +/- SE). Du ring blood sampling, no artifactual production of BK1-5 was detected for up to 60 s prior to denaturing the sample. This assay provides the first accu rate and precise method using MS to quantify BK1-5 in human blood as a mark er for the production of systemic bradykinin in humans. (C) 2001 Academic P ress.