Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, 1200 Cailun Rood, Shanghai 201203, China.
Institute of Cardiovascular Disease, Shuguang Hospital Affiliated with Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Shanghai 201203, China.
J Pharm Biomed Anal. 2018 May 10;153:175-181. doi: 10.1016/j.jpba.2018.02.045. Epub 2018 Feb 22.
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 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 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.
腺苷脱氨酶（ADA）是嘌呤核苷代谢中的关键酶，在多种疾病中发挥重要作用，如结核病、糖尿病、肝脏疾病和癌症。ADA 及其同工酶在体液中的活性测定在相关疾病的诊断和治疗中受到了广泛关注。以腺苷（AD）为底物的紫外光谱法是筛选潜在 ADA 抑制剂的经典方法，通过测量 265nm 处底物（AD）的减少或 248nm 处产物（肌苷）的增加来实现。然而，AD 和肌苷具有非常接近的最大吸收波长，并且该反应不确定，并且经常受到基质化合物或植物提取物背景颜色的干扰。因此，该方法通常会产生假阳性或假阴性结果。在这项研究中，开发了一种新颖、快速、灵敏和准确的超高效液相色谱-四极杆轨道阱高分辨率精确质量质谱（UHPLC-Q-Orbitrap HRMS）方法，通过直接测定 AD 的脱氨产物肌苷来测定和筛选 ADA 抑制剂。在沃特世 ACQUITY HSS T3 柱（2.1mm×100mm，1.8μm）上，采用等度洗脱（0.1%甲酸：甲醇=85:15，v/v），在 0.3mL/min 的流速下，在 2 分钟内实现了肌苷和氯代甲喹（内标）的适当分离，通过简单沉淀蛋白质。所开发方法的日内和日间精密度分别低于 7.17%和 8.99%。该方法具有小的总反应体积（60μL）、短的运行时间（2 分钟）、高灵敏度（肌苷的最低定量下限为 0.02μM）和低成本（ADA 的酶消耗为 0.007 单位/mL，AD 的底物为 3.74μM）的优点，并且没有基质效应（101.64%-107.12%）。稳定性结果表明，在所研究的条件下，所有分析物均稳定。该方法成功应用于检测中药中 ADA 的抑制活性。
