Bradykinin analysis revived – A validated method for determination of its stable metabolite in whole blood by LC–MS/MS

•A validated method for determination of the metabolite, bradykinin 1–5 (BK1–5), has been developed.•The validation confirms good linearity, repeatability (intra-run), reproducibility (inter-run), accuracy, and recovery.•Increased BK1–5 levels during hereditary angioedema attacks were measured. Investigation of bradykinin involvement in diseases like hereditary angioedema has been greatly hindered by its rapid metabolism and generation, induced by sampling. Because of this, reliable measurements of bradykinin have yet to be introduced in clinical practice. Prevention of bradykinin generation during sampling, and determination of the in vivo generated stable metabolite BK1–5, should allow a reliable indirect measure of bradykinin involvement. An LC–MS/MS method has been developed to determine BK1–5 in human whole blood. The method inactivates metabolizing enzymes with ethanol, followed by solid phase extraction (C18), separation (C8) and detection (linear ion trap) through the transitions 287.25→320.20 (y3, quantifier), 408.20 (b4, qualifier) for BK1–5, and 292.17→330.20 (y3, quantifier), 408.20 (b4, qualifier) for the heavy labelled internal standard. The method showed acceptable linearity (n=3, r2=0.994), intra-run precision (CV