Metabolomics Platform, Faculty of Biology and Medicine , University of Lausanne , 1005 Lausanne , Switzerland.
Vital-IT-Swiss Institute of Bioinformatics , 1015 Lausanne , Switzerland.
Anal Chem. 2019 Sep 17;91(18):11757-11769. doi: 10.1021/acs.analchem.9b02373. Epub 2019 Aug 26.
Acylcarnitines and amino acids are key players in energy metabolism; however, analytical methods for comprehensive and straightforward quantitative profiling of these metabolites, without derivatization or use of ion-pairing agents, are lacking. We therefore developed a hydrophilic interaction chromatography (HILIC)-based high-resolution mass spectrometry (HRMS) method for the simultaneous quantification of acylcarnitines and amino acids in a single run, while taking advantage of HRMS data acquired in full-scan mode to screen for additional derivatives and other polar metabolites. A single-step metabolite extraction with internal standard mixture (in methanol) warranted high-throughput sample preparation whose applicability was demonstrated on a panel of human biofluids (i.e., blood plasma, CSF, and urine) and brain tissue. Method accuracy was within 90-106% of validated NIST reference plasma concentrations for the panel of measured amino acids. Amino acid and acylcarnitine extraction recoveries were 87-100% on average, depending on the concentration range spiked. The coefficient of variation (CV) was 1-10% and 1-25% for intra- and interday measurements, respectively, with the highest CVs for the metabolites at the limit of quantification, depending on the biofluid. Acylcarnitine and amino acid signatures or chemical composition barcodes of the different biofluids and human brain tissue were acquired and biofluid- and tissue-associated differences were discussed in the context of their respective physiological roles. Significant differences were observed in the amino acid profiles, whereas acylcarnitine composition did not show biofluid-characteristic or brain region-specific pattern. The retrospective exploration of full-scan all-ion-fragmentation data allowed us to extract the information on unsaturated and hydroxylated acylcarnitine species, amines, and purine and pyrimidine metabolites. This merged targeted and untargeted approach provides an innovative strategy for simultaneous and comprehensive assessment of acylcarnitine and amino acid metabolism in clinical research studies using relevant biofluids and tissue extracts.
酰基肉碱和氨基酸是能量代谢的关键物质；然而，缺乏无需衍生化或使用离子对试剂即可全面、直接地定量分析这些代谢物的分析方法。因此，我们开发了一种基于亲水相互作用色谱（HILIC）的高分辨率质谱（HRMS）方法，可在单次运行中同时定量分析酰基肉碱和氨基酸，同时利用全扫描模式下获取的 HRMS 数据筛选出其他衍生物和其他极性代谢物。采用单步代谢物提取方法（用甲醇）和内标混合物，保证了高通量的样品制备，该方法在一系列人体生物流体（即血浆、CSF 和尿液）和脑组织中得到了验证。所测氨基酸的验证 NIST 参考血浆浓度的方法准确性在 90-106%之间。根据所加浓度范围，氨基酸和酰基肉碱的提取回收率平均为 87-100%。日内和日间测量的变异系数（CV）分别为 1-10%和 1-25%，取决于生物流体，最低定量限的代谢物的 CV 最高。获取了不同生物流体和人脑组织的酰基肉碱和氨基酸特征或化学组成条码，并讨论了其各自生理作用下的生物流体和组织差异。在氨基酸谱中观察到显著差异，而酰基肉碱组成没有显示出生物流体特征或脑区特异性模式。对全扫描全离子碎片数据的回顾性探索使我们能够提取不饱和和羟基化酰基肉碱、胺类、嘌呤和嘧啶代谢物的信息。这种合并的靶向和非靶向方法为使用相关生物流体和组织提取物在临床研究中同时全面评估酰基肉碱和氨基酸代谢提供了一种创新策略。
