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.

中文翻译：

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通过反相/离子交换质谱进行代谢谱分析。

代谢谱分析通常是通过质谱（MS）在反相（RP）和亲水作用色谱（HILIC）之后进行的，这些色谱独立进行，导致数据集重叠，或者以耦合方式进行，需要多个液相色谱（LC）系统。为了克服这些局限性，我们开发了使用在线RP离子交换（IEX）色谱柱排列和单个LC系统的20分钟色谱方法。这种配置可分离临床上重要的极性和非极性化合物，而无需衍生化或离子对试剂，从而可实现两种极性的电离。内部图书馆建立了397个真实标准品，包括酰基肉碱，氨基酸，胆汁酸，核苷，有机酸，类固醇激素和维生素。合并血浆和尿液样品的分析显示了5445和4111离子特征，分别导致了88和82个确认的代谢物鉴定。在临床相关浓度下以良好的精密度检测了代谢物，并证明了具有临床重要性的异构体（包括甲基丙二酸和琥珀酸，以及异亮氨酸和异亮氨酸/亮氨酸）具有良好的色谱分离效果。通过无监督主成分分析对样品进行评估显示出优异的分析质量。以及别异亮氨酸和异亮氨酸/亮氨酸。通过无监督主成分分析对样品进行评估显示出优异的分析质量。以及别异亮氨酸和异亮氨酸/亮氨酸。通过无监督主成分分析对样品进行评估显示出优异的分析质量。