Minkler Paul E, Stoll Maria S K, Ingalls Stephen T, Hoppel Charles L
Center for Mitochondrial Diseases, Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Center for Mitochondrial Diseases, Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA; Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Mol Genet Metab. 2017 Apr;120(4):363-369. doi: 10.1016/j.ymgme.2017.02.006. Epub 2017 Feb 8.
While selectively quantifying acylcarnitines in thousands of patient samples using UHPLC-MS/MS, we have occasionally observed unidentified branched-chain C8 acylcarnitines. Such observations are not possible using tandem MS methods, which generate pseudo-quantitative acylcarnitine "profiles". Since these "profiles" select for mass alone, they cannot distinguish authentic signal from isobaric and isomeric interferences. For example, some of the samples containing branched-chain C8 acylcarnitines were, in fact, expanded newborn screening false positive "profiles" for medium-chain acyl-CoA dehydrogenase deficiency (MCADD). Using our fast, highly selective, and quantitatively accurate UHPLC-MS/MS acylcarnitine determination method, we corrected the false positive tandem MS results and reported the sample results as normal for octanoylcarnitine (the marker for MCADD). From instances such as these, we decided to further investigate the presence of branched-chain C8 acylcarnitines in patient samples. To accomplish this, we synthesized and chromatographically characterized several branched-chain C8 acylcarnitines (in addition to valproylcarnitine): 2-methylheptanoylcarnitine, 6-methylheptanoylcarnitine, 2,2-dimethylhexanoylcarnitine, 3,3-dimethylhexanoylcarnitine, 3,5-dimethylhexanoylcarnitine, 2-ethylhexanoylcarnitine, and 2,4,4-trimethylpentanoylcarnitine. We then compared their behavior with branched-chain C8 acylcarnitines observed in patient samples and demonstrated our ability to chromographically resolve, and thus distinguish, octanoylcarnitine from branched-chain C8 acylcarnitines, correcting false positive MCADD results from expanded newborn screening.
在使用超高效液相色谱-串联质谱法（UHPLC-MS/MS）对数千份患者样本中的酰基肉碱进行选择性定量分析时，我们偶尔会观察到未识别的支链C8酰基肉碱。使用串联质谱法无法进行此类观察，因为串联质谱法只能生成伪定量的酰基肉碱“谱图”。由于这些“谱图”仅根据质量进行选择，所以无法区分真实信号与等压和同分异构干扰。例如，一些含有支链C8酰基肉碱的样本实际上是中链酰基辅酶A脱氢酶缺乏症（MCADD）扩大新生儿筛查的假阳性“谱图”。使用我们快速、高选择性且定量准确的UHPLC-MS/MS酰基肉碱测定方法，我们纠正了假阳性的串联质谱结果，并将样本结果报告为辛酰肉碱（MCADD的标志物）正常。从这些实例中，我们决定进一步研究患者样本中支链C8酰基肉碱的存在情况。为实现这一目标，我们合成并通过色谱法表征了几种支链C8酰基肉碱（除丙戊酰肉碱外）：2-甲基庚酰肉碱、6-甲基庚酰肉碱、2,2-二甲基己酰肉碱、3,3-二甲基己酰肉碱、3,5-二甲基己酰肉碱、2-乙基己酰肉碱和2,4,4-三甲基戊酰肉碱。然后，我们将它们的行为与在患者样本中观察到的支链C8酰基肉碱进行了比较，并证明了我们能够通过色谱法分离，从而区分辛酰肉碱和支链C8酰基肉碱，纠正扩大新生儿筛查中MCADD的假阳性结果。
