Automated assay for the determination of methylmalonic acid, total homocysteine, and related amino acids in human serum or plasma by means of methylchloroformate derivatization and gas chromatography-mass spectrometry
Automated assay for the determination of methylmalonic acid, total homocysteine, and related amino acids in human serum or plasma by means of methylchloroformate derivatization and gas chromatography-mass spectrometry
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DOI：
10.1373/clinchem.2005.053835
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发表时间：
2005-11-01
影响因子：
9.3
通讯作者：
Ueland, PM
中科院分区：
文献类型：
--
作者：
Windelberg, A;Årseth, O;Ueland, PM
背景：血清或血浆中甲基丙二酸(MMA)和总同型半胱氨酸(THcy)的联合检测有助于诊断和鉴别钴胺和叶酸缺乏。建立并验证了一种采用自动进样的同位素稀释气相色谱-质谱仪(GC-MS)测定血清或血浆中MMA、tHcy及相关氨基酸Met、总半胱氨酸(TCys)、丝氨酸(Ser)和甘氨酸(Gly)的方法。方法：血清或血浆样品经二硫代赤藓糖醇处理，乙醇去蛋白，氯甲酸甲酯和甲苯一步衍生化提取。所有液体处理由机器人工作站在96孔(1毫升)微滴定板中进行。结果：检出限(信噪比，5：1)在0.03mU/L(甲基丙烯酸甲酯)~10mU/L(丝氨酸，t-半胱氨酸)之间。MMA和tHcy的线性范围为100mU/L，蛋氨酸、tCys、Ser和Gly的线性范围为1000mU/L。日内变异系数为0.7%~3.6%(n=20)，日间变异系数为2.1%~8.1%(n=20)。不同分析方法的回收率在79%~99%之间。结论：该方法将样品前处理与GC-MS分析相结合，方法简便、自动化，适用于MMA、tHcy及相关氨基酸的联合测定。(C)2005年美国临床化学协会。
Background: The combined measurement of methylmalonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and distinguishing between cobalamin and folate deficiencies. We developed and validated an isotope-dilution gas chromatography-mass spectrometry (GC-MS) method with automated sample workup for the determination of MMA, tHcy, and the related amino acids Met, total cysteine (tCys), Ser, and Gly in serum or plasma.Methods: Serum or plasma samples (100 mu L) were treated with a reductant (dithioerythritol), deproteinized with ethanol, and derivatized and extracted in a single step by the addition of methylchloroformate and toluene. All liquid handling was performed in 96-well (1 mL) microtiter plates by a robotic workstation. The N(S)-methoxycarbonyl ethyl ester derivatives were analyzed by GC-MS in the selected-ion monitoring mode.Results: Detection limits (signal-to-noise ratio, 5:1) were between 0.03 mu mol/L (MMA) and 10 mu mol/L (Ser, tCys). The assay was linear to 100 mu mol/L for MMA and tHcy and to 1000 mu mol/L for Met, tCys, Ser, and Gly. The within-day CVs ranged from 0.7% to 3.6% (n = 20), and the between-day CVs from 2.1% to 8.1% (n = 20). The recovery was between 79% and 99% for the different analytes.Conclusion: This assay combines a simple and automated sample preparation with selective and sensitive GC-MS analysis and is well suited for the combined measurement of MMA, tHcy, and the related amino acids. (c) 2005 American Association for Clinical Chemistry.