Rapid determination of C4-acylcarnitine and C5-acylcarnitine isomers in plasma and dried blood spots by UPLC-MS/MS as a second tier test following flow-injection MS/MS acylcarnitine profile analysis
Rapid determination of C4-acylcarnitine and C5-acylcarnitine isomers in plasma and dried blood spots by UPLC-MS/MS as a second tier test following flow-injection MS/MS acylcarnitine profile analysis
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DOI：
10.1016/j.ymgme.2010.05.012
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发表时间：
2010-09-01
影响因子：
3.8
通讯作者：
Sweetman, Lawrence
中科院分区：
文献类型：
--
作者：
Forni, Sabrina;Fu, Xiaowei;Sweetman, Lawrence
背景：大多数新生儿筛查和生化遗传学实验室常规采用流动注射 MS/MS 方法检测酰基肉碱升高情况；然而，该技术无法区分同量异位化合物；因此，需要色谱分离来定量异构体，以鉴别诊断某些先天性代谢缺陷。 方法：开发了一种 UPLC-MS/MS 方法，用于同时定量异丁酰肉碱和丁酰肉碱，以及另一种 UPLC-MS/MS 方法，用于定量异戊酰肉碱、(S) 和 (R) 2-甲基丁酰肉碱、新戊酰肉碱和戊酰肉碱。用甲醇提取血浆和干燥的血斑样品并用丁醇HCl衍生化。氘标记的内标用于定量。使用甲醇/水梯度，使用 C18 BEH，1 x 100 mm，1.7 μm UPLC 柱，在 60°C 下进行分离；运行时间少于10分钟。使用 Quattro Premier 三重四极杆在正离子模式下进行电喷雾电离来检测异构体。结果：丁酰肉碱和异丁酰肉碱的血浆和干血斑的日内精密度分别为 1.4% 至 14%，准确度分别为 88% 至 114%。在血浆或干血斑中，C5-酰基肉碱异构体的精确度分别为 1.3% 至 15%，准确度为 87% 至 119%。异丁酰肉碱和丁酰肉碱各浓度的日间精度均在 20% 以内。浓度高于正常范围的 2-甲基丁酰肉碱和异戊酰肉碱的精确度在 24% 以内。结论：基于通过 UPLC-MS/MS 分离 C4-酰基肉碱和 C5-酰基肉碱异构体的两种诊断测试，可快速鉴别诊断 SCAD 缺陷与 IBCD 缺陷以及 IVA 与 2-MBCD 缺陷。 C5-酰基肉碱的分离可以揭示含新戊酸抗生素引起的假性升高。证实了母体产生新戊酸前药抗生素导致的新生儿筛查结果异常。这种异构体的分离可以解决新生儿筛查和代谢测试结果不明确的情况下的多种诊断挑战。 (C) 2010 Elsevier Inc. 保留所有权利。
Background: Flow-injection MS/MS methods for elevated acylcarnitines are routinely performed in most newborn screening and biochemical genetics laboratories; however this technique cannot distinguish between isobaric compounds; therefore, chromatographic separation is required to quantitate isomers for differential diagnosis of some inborn errors of metabolism.Methods: A UPLC-MS/MS method has been developed for the simultaneous quantitation of isobutyrylcarnitine and butyrylcarnitine, and a second UPLC-MS/MS method for the quantitation of isovalerylcarnitine, (S) and (R) 2-methylbutyrylcarnitine, pivaloylcarnitine and valerylcarnitine. Plasma and dried blood spots samples are extracted with methanol and derivatized with butanolic HCI. Deuterium labeled internal standards are used for quantitation. Separation is obtained using a methanol/water gradient with a C18 BEH, 1 x 100 mm, 1.7 mu m UPLC column, at 60 degrees C; run time is less than 10 min. The isomers are detected with a Quattro Premier triple quadrupole, with electrospray ionization in positive ion mode.Results: Intra-day precision in plasma and dried blood spots ranged from 1.4% to 14% and accuracy from 88% to 114% respectively for butyrylcarnitine and isobutyrylcarnitine. Precision for the isomers of C5-acylcarnitine ranged from 1.3% to 15% and accuracy 87% to 119%, respectively in plasma or dried blood spots. Inter-day precision was within 20% at each concentration of isobutyrylcarnitine and butyrylcarnitine. Precision for 2-methylbutyrylcarnitine and isovalerylcarnitine at concentrations above the normal range was within 24%.Conclusions: Two diagnostic tests based on the separation of C4-acylcarnitine and C5-acylcarnitine isomers by UPLC-MS/MS provide fast differential diagnosis of SCAD deficiency versus IBCD deficiency and IVA versus 2-MBCD deficiency. The separation of C5-acylcarnitines can reveal false elevation due to pivalic acid-containing antibiotics. Abnormal newborn screen results due to pivalate-generating prodrug antibiotics of maternal origin were confirmed. This separation of isomers can resolve multiple diagnostic challenges in both newborn screening and in cases with ambiguous metabolic test results. (C) 2010 Elsevier Inc. All rights reserved.