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.