High-performance liquid chromatographic determination of bradykinin in saliva: a critical review and a new method

Because of difficulties or dubious results with previously published methodologies, a new semi-automated HPLC method with UV absorbance detection was developed and applied to the determination of bradykinin (BK) in human saliva. The new method consisted of an uncomplicated sample preparation involving the addition to saliva of an equal volume of 0.1 M orthophosphoric acid to stabilize BK, vortex-mixing, centrifugation, and separation, followed by chromatography of the supernatant phase on a C8, 150×3.9-mm (I.D.) stainless steel column. The mobile phase was composed of 19% acetonitrile/0.1% trifluoroacetic acid at flow-rate of 0.4 ml/min. Using UV detection at 220 nm, the detection limit was 1 ng/ml for the BK standard, and 7 ng/ml for the assay of endogenous salivary BK. The orthophosphoric acid initially added to the saliva allowed BK to be stabilized from enzymic degradation at 20°C for 5 days and at 4°C for 60 days. Assignment made to the peak with the chromatographic properties of salivary BK was confirmed by HPLC–MS with an electrospray interface. This paper presents a new method that is reproducible, reliable and allows kinetic studies of salivary BK to be performed for clinical investigations.