Measurement of plasma renin activity with use of HPLC-electrospray- tandem mass spectrometry

Abstract

Background: The measurement of renin activity is complicated by difficulties in the quantification of angiotensin 1 (Ang1), the product of the renin-catalyzed reaction. We report an HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of Ang1 as a measure of plasma renin activity (PRA). Methods: After incubation (37 °C for 3 or 18 h), samples were prepared using C18 solid-phase extraction. [Val]5Ang1 was used as the internal standard (IS). Chromatography was performed on a C18 column, using 200 mL/L ammonium acetate buffer-800 mL/L methanol as the mobile phase. The flow rate was 150 μL/min, with a chromatographic run time of 5 min/sample. Mass spectrometric detection was in the positive ionization mode with selected reaction monitoring (Ang1 m/z 649.0→784.0; IS m/z 641.9→770.4). Results: The assay was linear over the range 2.5-500 ng Ang1/mL, which corresponded to a limit of detection (signal- to-noise ratio of 3:1) of PRA of 0.14 ng Ang1 · mL-1 · h-1. The imprecision (CV) of the assay at PRA values of 26.1, 13.5, 3.2, and 0.78 ng Ang1 · mL-1 · h-1 was 7.0%, 7.0%, 15%, and 11%, respectively. Absolute recoveries were 92.3% (Ang1) and 87.4% (IS). Incubation times of 3 h vs 18 h in the PRA assay gave good agreement at PRA <2 ng Ang1 · mL-1 · h-1, but samples with a PRA of 2-5 ng Ang1 · mL-1 · h-1 gave lower PRA results after incubation for 18 h than after 3 h. We compared the HPLC-ESI- MS/MS assay and an RIA for the determination of PRA, with PRA incubation times of 3 h and 1.5 h, respectively. The mean PRA based on RIA of Ang1 was higher than that obtained using HPLC-ESI-MS/MS. Conclusion: The HPLC-ESI- MS/MS method allows sensitive and specific measurement of PRA. The higher activities measured with the RIA method highlight its potential for overestimation of PRA.