Quantification of BK1-5, the Stable Bradykinin Plasma Metabolite in Humans, by a Highly Accurate Liquid-Chromatographic Tandem Mass Spectrometric Assay

Abstract

Bradykinin is a vasoactive nonapeptide involved in cardiorenal physiology and inflammatory states. It has been linked to the pathophysiology of hypertension and diabetes. Correlating levels of bradykinin with disease states has been hampered by its rapid degradation, artifactual production during blood sampling, and nonspecific radioimmunoassay techniques. We previously identified BK1-5 as the stable in vivo plasma metabolite of systemic bradykinin in humans. We now report a sensitive and specific assay method for BK1-5 in human blood utilizingliquid chromatography–tandem mass spectrometry(MS) with electrospray ionization. [13C2,15N]Glycine was incorporated into chemically synthesized BK1-5 for use as an internal standard. Blood samples (5 ml) were collected into 15-ml chilled ethanol to prevent artifactual kinin production and degradation. BK1-5 in ethanolic plasma supernatant was purified on a polymeric solid phase extraction cartridge. MS analysis was in 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). During 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 accurate and precise method using MS to quantify BK1-5 in human blood as a marker for the production of systemic bradykinin in humans.