We report a selective LC–MS/MS method for the simultaneous quantitative determinations of the adenosine A2a receptor antagonist NIR178 (NIR178) and its major metabolite NJI765 in human plasma. Sample preparation steps involved protein precipitation, sample evaporation and reconstitution using a plasma sample volume of 0.1 ml plasma. Separation was achieved in 10 min on an Acquity UPLC BEH C₁₈ 1.7 μm, 2.1 × 50 mm column heated at 60°C with a gradient elution at 0.6 ml/min mobile phase made of water and acetonitrile both acidified with 0.1% formic acid. The detection was performed in positive ion mode and quantification based on multiple reaction monitoring. The linear response range was 1.00–1,000 ng/ml using a 1/x² weighting factor. The intra‐ and inter‐day accuracies (bias %) and intra‐ and inter‐day precisions (CV, %) obtained for NIR178 and NJI765 were within the acceptance criteria. The normalized NIR178 and NJI765 matrix factor calculated from six lots of normal, lipemic and hemolyzed plasmas ranged from 0.97 to 1.05. The normalized recoveries of both NIR178 and NJI765 compared with their internal standards were consistent and reproducible with a CV ≤8.0. This method was successfully applied to support pharmacokinetic studies in adult patients with cancer.

中文翻译：

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开发和验证用于人血浆中腺苷A2a受体拮抗剂NIR178及其单羟基代谢物的定量分析的LC-MS / MS方法：在临床药代动力学中的应用。

我们报告了一种选择性LC-MS / MS方法，用于同时定量测定人血浆中腺苷A2a受体拮抗剂NIR178（NIR178）及其主要代谢物NJI765。样品制备步骤涉及蛋白质沉淀，样品蒸发和使用0.1 ml血浆的血浆样品重建。在60°C加热的Acquity UPLC BEH C ₁₈ 1.7μm，2.1×50 mm色谱柱上，于10分钟内完成分离，分离度为0.6 ml / min，由水和乙腈制成的流动相均用0.1％甲酸酸化。检测以阳离子模式进行，并基于多反应监测进行定量。线性响应范围是1.00–1,000 ng / ml，使用1 / x ²加权因子。NIR178和NJI765的日内和日间精度（偏差％）以及日内和日间精度（CV，％）均在接受标准之内。从六批正常，脂血和溶血血浆中计算出的归一化NIR178和NJI765矩阵因子在0.97至1.05之间。与内标相比，NIR178和NJI765的归一化回收率一致且可重复，CV≤8.0。该方法已成功地用于支持成人癌症患者的药代动力学研究。