Adenosine deaminase (ADA), which is a key enzyme in the metabolism of purine nucleosides, plays important roles in diverse disorders, such as tuberculosis, diabetes, liver disorders, and cancer. Determination of the activities of ADA and its isoenzymes in body fluids has received considerable attention in the diagnosis and treatment of relative diseases. Ultraviolet spectroscopy with adenosine (AD) as a substrate is a classical approach for screening potential ADA inhibitors by measuring the decrease in substrate (AD) at 265 nm or increase in the product (inosine) at 248 nm. However, AD and inosine share a very close maximum absorption wavelength, and the reaction is uncertain and is frequently interfered by the background color of matrix compounds or plant extracts. Thus, the method usually yields false positive or negative results. 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 x 100 mm, 1.8 mu m) 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 4), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02 mu M for inosine), and low cost (small enzyme consumption of 0.007 unit mL(-1) for ADA and substrate of 3.74 mu M 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. (C) 2018 Elsevier B.V. All rights reserved.
[1]Shanghai Univ Tradit Chinese Med, Inst Chinese Mat Med, MOE Key Lab Standardizat Chinese Med, 1200 Cailun Rood, Shanghai 201203, Peoples R China.
[2]SATCM Key Lab New Resources & Qual Evaluat Chines, 1200 Cailun Rood, Shanghai 201203, Peoples R China.
[3]Shanghai R&D Ctr Standardizat Chinese Med, 199 Guoshoujing Rd, Shanghai 201203, Peoples R China.
[4]Shanghai Univ Tradit Chinese Med, Inst Liver Dis, Shuguang Hosp, Key Lab Liver & Kidney Dis,Minist Educ, 528 Zhangheng Rd, Shanghai 201203, Peoples R China.
[5]Shanghai Univ Tradit Chinese Med, Shuguang Hosp, Inst Cardiovasc Dis, 528 Zhangheng Rd, Shanghai 201203, Peoples R China.
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