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中的临床相关标志物,丙酸盐,和钴胺素代谢。本研究旨在建立一种超高效液相色谱-串联质谱(UPLC-MS/MS)同时测定同型半胱氨酸的方法,蛋氨酸,甲基丙二酸和2-甲基柠檬酸在干血斑点。从每个校准器上冲压出三个3.2mm的圆盘,质量控制,并将干血点取样到96孔U形板中。每个样品用内标加标并提取。然后将上清液转移到另一个96孔U形板中。氮气干燥后,干燥的残留物被重建,离心,并将得到的上清液转移到另一个96孔板进行分析。该方法使用UPLC-MS/MS在3分钟内进行,根据指导文件进行验证,并应用于72例蛋氨酸确诊患者的样本,丙酸盐,和钴胺素代谢紊乱.UPLC-MS/MS方法提供了四种分析物的令人满意的分离。所有分析物的R2值≥0.9937。回收率在94.17-114.29%之间,日内和日间精度的变异系数分别为0.19%至5.23%和1.02%至6.89%,分别。对于四种分析物没有检测到显著的携带,大多数证实的样本表现出相关疾病的生物标志物模式特征。成功开发了一种简单快速的UPLC-MS/MS方法,已验证,并应用于临床样品中同时测定总同型半胱氨酸,蛋氨酸,甲基丙二酸,和2-甲基柠檬酸在干血斑点。
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 R2 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.