Absolute quantification of endogenous angiotensin II levels in human plasma using ESI-LC-MS/MS

Abstract

Background: Angiotensin II acts as a peptide hormone and component of renin-angiotensin- system (RAS) regulating the blood pressure, and seems to be involved in renal and vascular disorders. There is no reliable quantification method for angiotensin II available until now and the angiotensin II plasma levels described in the literature are correspondingly strongly divergent. Therefore, we developed and validated a sensitive, selective and reliable LC-ESI-MS/MS method for absolute quantification of angiotensin II concentration in human plasma based on the AQUA strategy. Methods: Plasma samples were extracted using MAX Oasis cartridges and were subjected to a further immunoaffinity-purification using immobilized anti-angiotensin II antibodies in order to isolate endogenous angiotensin II. Stable isotope (13C- and 15∈N-) labeled angiotensin II was used as an internal standard. The fractionated samples were analysed using LC-ESI-MS/MS. Results: The calibration curve was established in plasma in the concentration range 6-240 pM (r2∈>∈0.999). The developed and validated method was successfully applied for quantification of endogenous angiotensin II in human plasma of healthy volunteers and chronic kidney disease (CKD-5D) patients. The mean plasma angiotensin II levels were found to be 18.4∈±∈3.3 pM in healthy subjects and 64.5∈±∈32.4 pM in CKD-5D patients (each n =9). Conclusion: The LC-ESI-MS/MS-based method for quantification of angiotensin II levels in human plasma was successfully evaluated within the study. This method is applicable for clinical applications aiming at the validation of the impact of highly physiologically and pathophysiologically active angiotensin II.