Rapid, reliable, and sensitive detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS and its application to inhibitory activity evaluation of traditional Chinese medicines

In this study, a novel, rapid, sensitive, and accurate ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method was developed for determining and screening ADA inhibitors by directly determining the deamination product of AD, inosine. A proper separation was achieved for inosine and chlormequat (internal standard) within 2 min via isocratic elution (0.1% formic acid:methanol = 85:15, v/v) at a flow rate of 0.3 mL min−1 on a Waters ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 μm) following a simple precipitation of proteins. The intra- and inter-day precisions of the developed method were below 7.17% and 8.99%, respectively. The method exhibited advantages of small total reaction volume (60 μL), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02 μM for inosine), and low cost (small enzyme consumption of 0.007 unit mL−1 for ADA and substrate of 3.74 μM for AD in individual inhibition), and no matrix effects (101.64%–107.12%). Stability results showed that all analytes were stable under the investigated conditions. The developed method was successfully applied to the detection of the inhibitory activity of ADA from traditional Chinese medicines. Graphical abstract
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research