Method Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry for Angiotensin-II in Human Plasma: Application to Study Interaction Between Atorvastatin & Olmesartan Drug Combination

Abstract

Simple and sensitive Liquid Chromatography-Tandem Mass Spectrometry (LCMS/MS) method was developed and validated, then was implicated on hypertensive human subjects to study drug interaction of atorvastatin (ATVS) and Olmesartan (OLM) on status of Angiotensin-II (ANG-II). The ANG-II in plasma was extracted with 5mL methanol containing 5% formic acid through C18 (cartridges) liquid-liquid extraction, dried and reconstituted with 1mL of 16% acetonitrile in 0.1% formic acid in water. The chromatographic separation of ANG-II with a Agilent technology 6410 Triple quadrupole was carried multiple reaction monitoring scan mode with a Agilent 1290 Infinity LC system for UHPLC. The sample were separated on a (Thermo Scientific) Hy-Purity advance (50×4.6mm, 5μm) using Mobile Phase A: 16% acetonitrile in 0.1% formic acid in water and Mobile Phase B: 0.1% formic acid in methanol at a flow rate of 0.3mL/min, performed at ambient temperature. The mobile phase gradient of 16% acetonitrile in water was linearly increased to 38% acetonitrile over 10min and subsequently the mobile-phase was increased to 100% acetonitrile over 15min. The developed method was validated for specificity, accuracy, precision, stability, linearity, sensitivity and recovery. The method was linear between peak area ratio of standard and internal standard over the range of 50-800ng/mL. The method was successfully applied for the drug interaction study revealed levels of ANG-II were significantly higher in ATVS+OLM treatment condition as compared to individual treatment of OLM. This reflects the reason of low effectiveness of ATVS+OLM in combination instead of synergistic activity.