Merged Targeted Quantification and Untargeted Profiling for Comprehensive Assessment of Acylcarnitine and Amino Acid Metabolism

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（acylcarnitines）与氨基酸是能量代谢的核心参与者。然而，目前尚缺乏无需衍生化处理或使用离子对试剂，即可对这类代谢物进行全面且简便的定量表征的分析方法。为此，本研究开发了一种基于亲水作用色谱法（hydrophilic interaction chromatography, HILIC）结合高分辨质谱法（high-resolution mass spectrometry, HRMS）的分析方法，可在单针进样中同时定量酰基肉碱与氨基酸；同时利用全扫描模式下采集的HRMS数据，筛选额外衍生产物及其他极性代谢物。采用含内标混合液（甲醇溶剂）的单步代谢物提取流程，可实现高通量样品前处理；该方法的适用性通过一组人体生物体液（即血浆、脑脊液（cerebrospinal fluid, CSF）和尿液）及脑组织样本得到验证。针对所检测的氨基酸，方法准确度处于经美国国家标准与技术研究院（National Institute of Standards and Technology, NIST）验证的血浆参考浓度的90%~106%区间内。氨基酸与酰基肉碱的提取回收率平均为87%~100%，具体数值取决于加标浓度区间。批内与日间检测的变异系数（CV）分别为1%~10%和1%~25%，其中处于定量限的代谢物的变异系数最高，且该情况因生物体液类型而异。获取了不同生物体液及人体脑组织的酰基肉碱与氨基酸特征谱（即化学成分条形码），并结合各自的生理功能探讨了生物体液与组织间的相关差异。研究发现氨基酸谱存在显著差异，而酰基肉碱组成未呈现出生物体液特异性或脑区特异性模式。对全扫描全离子碎裂数据的回顾性分析，使我们得以提取不饱和、羟基化酰基肉碱物种、胺类以及嘌呤和嘧啶类代谢物的相关信息。这种融合靶向与非靶向的分析策略，为利用相关生物体液及组织提取物开展临床研究中同时全面评估酰基肉碱与氨基酸代谢提供了创新方案。