Department of Genetics and Metabolism, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
School of Health in Social Science, The University of Edinburg, United Kingdom.
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Aug 15;1244:124253. doi: 10.1016/j.jchromb.2024.124253. Epub 2024 Jul 24.
Homocysteine, methionine, methylmalonic acid and 2-methylcitric acid are clinically relevant markers in the methionine, propionate, and cobalamin metabolism. This study aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneously determining total homocysteine, methionine, methylmalonic acid and 2-methylcitric acid in dried blood spots. Three 3.2 mm discs were punched from each calibrator, quality control, and sample dried blood spot into a 96-well U-plate. Each sample was spiked with internal standards and extracted. Then the supernatant was transferred to another 96-well U-plate. After nitrogen drying, the dried residues were reconstituted, centrifuged, and the resulting supernatant was transferred to another 96-well plate for analysis. The method was performed using UPLC-MS/MS within 3 min, validated according to guidance documents, and applied to 72 samples from confirmed patients with methionine, propionate, and cobalamin metabolism disorders. The UPLC-MS/MS method provided satisfactory separation of the four analytes. The R values were ≥ 0.9937 for all analytes. The recoveries ranged from 94.17 to 114.29 %, and the coefficients of variation for intraday and interday precision were 0.19 % to 5.23 % and 1.02 % to 6.89 %, respectively. No significant carry-over was detected for the four analytes, and most of confirmed samples exhibited biomarker patterns characteristic of the relevant disorders. A simple and fast UPLC-MS/MS method was successfully developed, validated, and applied to clinical samples for the simultaneous determination of total homocysteine, methionine, methylmalonic acid, and 2-methylcitric acid in dried blood spots.
同型半胱氨酸、蛋氨酸、甲基丙二酸和 2-甲基柠檬酸是蛋氨酸、丙酸和钴胺素代谢中具有临床相关性的标志物。本研究旨在开发和验证一种超高效液相色谱-串联质谱（UPLC-MS/MS）方法，用于同时测定干血斑中的总同型半胱氨酸、蛋氨酸、甲基丙二酸和 2-甲基柠檬酸。从每个校准品、质控品和样本的干血斑中用 3.2mm 打孔器打出 3 个 3.2mm 圆片，放入 96 孔 U 型板中。每个样本加入内标后进行提取。然后将上清液转移到另一个 96 孔 U 型板中。氮吹干燥后，将干燥残渣重新溶解，离心，将得到的上清液转移到另一个 96 孔板中进行分析。该方法在 3 分钟内完成 UPLC-MS/MS 分析，根据指导文件进行验证，并应用于 72 例确诊的蛋氨酸、丙酸和钴胺素代谢紊乱患者的样本。UPLC-MS/MS 方法提供了对 4 种分析物的满意分离。所有分析物的 R 值均≥0.9937。回收率范围为 94.17%至 114.29%，日内和日间精密度的变异系数分别为 0.19%至 5.23%和 1.02%至 6.89%。未检测到 4 种分析物的明显交叉污染，大多数确诊样本显示出与相关疾病特征一致的生物标志物模式。成功开发、验证并应用于临床样本的简单快速的 UPLC-MS/MS 方法，用于同时测定干血斑中的总同型半胱氨酸、蛋氨酸、甲基丙二酸和 2-甲基柠檬酸。
