Department of Pathology, Stanford University School of Medicine, 3375 Hillview Ave, Palo Alto, CA 94304, USA.
Clinical Biochemical Genetics Laboratory, Stanford Health Care, 3375 Hillview Ave, Palo Alto, CA 94304, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 15;1143:122072. doi: 10.1016/j.jchromb.2020.122072. Epub 2020 Mar 16.
Metabolic profiling is commonly achieved by mass spectrometry (MS) following reversed-phase (RP) and hydrophilic interaction chromatography (HILIC) either performed independently, leading to overlapping datasets, or in a coupled configuration, requiring multiple liquid chromatography (LC) systems. To overcome these limitations, we developed a single, 20-minute chromatographic method using an in-line RP-ion-exchange (IEX) column arrangement and a single LC system. This configuration separates clinically significant polar and non-polar compounds without derivatization or ion-pairing reagents, allowing ionization in both polarities. An in-house library was created with 397 authentic standards, including acylcarnitines, amino acids, bile acids, nucleosides, organic acids, steroid hormones, and vitamins. Analysis of pooled plasma and urine samples revealed 5445 and 4111 ion features, leading to 88 and 82 confirmed metabolite identifications, respectively. Metabolites were detected at clinically relevant concentrations with good precision, and good chromatographic separation was demonstrated for clinically significant isomers including methylmalonic acid and succinic acid, as well as alloisoleucine and isoleucine/leucine. Evaluation of the samples by unsupervised principal component analysis showed excellent analytical quality.
代谢组学通常通过质谱 (MS) 进行分析，采用反相 (RP) 和亲水相互作用色谱 (HILIC)，要么独立进行，导致数据集重叠，要么采用耦合配置，需要多个液相色谱 (LC) 系统。为了克服这些限制，我们开发了一种单一的、20 分钟的色谱方法，采用在线 RP-离子交换 (IEX) 柱排列和单个 LC 系统。这种配置可分离临床意义重大的极性和非极性化合物，无需衍生化或离子对试剂，允许在两种极性下进行电离。使用 397 种真实标准品创建了一个内部库，包括酰基肉碱、氨基酸、胆汁酸、核苷、有机酸、甾体激素和维生素。对混合血浆和尿液样本的分析显示出 5445 和 4111 个离子特征，分别导致 88 和 82 种确认的代谢物鉴定。代谢物以临床相关浓度检测，具有良好的精密度，并且对于临床意义重大的异构体（包括甲基丙二酸和琥珀酸，以及异亮氨酸和亮氨酸/异亮氨酸）具有良好的色谱分离。通过无监督主成分分析对样品进行评估，显示出出色的分析质量。
