Department of Laboratory Medicine, The First Hospital of Jilin University, Changchun 130061, China.
Genetic Diagnosis Center, The First Hospital of Jilin University, Changchun 130061, China.
Molecules. 2022 Nov 7;27(21):7619. doi: 10.3390/molecules27217619.
A simple sample preprocessing method was developed for the quantitative determination of amantadine (AMT) in human plasma by liquid chromatography-tandem mass spectrometry cubed (LC-MS3). The LC-MS3 system comprised a Shimadzu Exion LC-20AD HPLC pump coupled with a QTRAP 5500 mass spectrometer. First, the plasma samples were pretreated using acetonitrile as the extracting solution to precipitate protein. Next, amantadine and amantadine-d15 (AMT-d15) were separated on an Agilent Poroshell 120 SB-C18 column (4.6 mm × 50 mm, 2.7 μm) using isocratic elution with solvent A (70% 0.1% formic acid) and solvent B (30% acetonitrile) at a flow rate of 0.8 mL/min. The total run time for each sample was 3 min. The system used triple-stage fragmentation transitions at m/z 152.2→135.3→107.4 for AMT quantification in the positive ion mode and m/z 167.0→150.3→118.1 for AMT-d15 quantification. The LC-MS3 assay was linear (r > 0.995) with a concentration range of 50−1500 ng/mL. The lower limit of quantification (LLOQ) was 50 ng/mL, and the intra-day and inter-day accuracies and precisions were less than 8.0% at all concentrations. In addition, the recoveries and matrix effect for AMT in human plasma were within acceptable limits. In terms of stability, AMT had no significant degradation under all conditions. All the results met the requirements of the guidelines of the Food and Drug Administration (FDA) for biological method validation. The novelty of the MS3 assay was that it presented a methodology with higher selectivity and sensitivity. This method was successfully applied to 44 human plasma samples, and the obtained quantitative results were compared with another liquid chromatography-multiple reaction monitoring (LC-MRM) method. The Passing-Bablok regression coefficients and Bland-Altman plot revealed no difference between the LC-MS3 and LC-MRM methods, implying that the developed LC-MS3 method is a reliable and accurate assay for AMT determination in human plasma. These results are also a proof of concept for determining chemicals in biological samples by the LC-MS3 strategy.
建立了一种简单的样品预处理方法，用于通过液相色谱-串联质谱法（LC-MS3）定量测定人血浆中的金刚烷胺（AMT）。LC-MS3 系统包括岛津 Exion LC-20AD HPLC 泵，与 QTRAP 5500 质谱仪耦合。首先，使用乙腈作为提取溶液沉淀蛋白质来预处理血浆样品。然后，在 Agilent Poroshell 120 SB-C18 柱（4.6mm×50mm，2.7μm）上，使用溶剂 A（70%0.1%甲酸）和溶剂 B（30%乙腈）进行等度洗脱，流速为 0.8mL/min，分离金刚烷胺和金刚烷胺-d15（AMT-d15）。每个样品的总运行时间为 3min。系统在正离子模式下使用三重碎裂转换，m/z 152.2→135.3→107.4 用于 AMT 定量，m/z 167.0→150.3→118.1 用于 AMT-d15 定量。LC-MS3 测定法呈线性（r＞0.995），浓度范围为 50-1500ng/mL。定量下限（LLOQ）为 50ng/mL，日内和日间精密度和准确度在所有浓度下均小于 8.0%。此外，金刚烷胺在人血浆中的回收率和基质效应均在可接受范围内。就稳定性而言，金刚烷胺在所有条件下均无明显降解。所有结果均符合食品和药物管理局（FDA）生物方法验证指南的要求。MS3 测定法的新颖之处在于它提供了一种具有更高选择性和灵敏度的方法。该方法成功应用于 44 个人血浆样品，并将获得的定量结果与另一种液相色谱-多重反应监测（LC-MRM）方法进行比较。Passing-Bablok 回归系数和 Bland-Altman 图表明 LC-MS3 法与 LC-MRM 法之间无差异，这表明所开发的 LC-MS3 法是一种可靠且准确的测定人血浆中 AMT 的方法。这些结果也是通过 LC-MS3 策略测定生物样品中化学物质的概念验证。
