Targeted amino acid and related amines analysis based on iTRAQ®-LC-MS/MS for discovering potential hepatotoxicity biomarkers.

Drug induced liver injury (DILI) is a diverse set of liver injury occurring after exposure to any manufactured or naturally occurring chemical compounds. DILI has already become the leading cause of acute liver failure in developed countries. Early diagnosis is of great significance for the prevention and treatment of DILI, which can effectively avoid its progress to acute liver failure. In this paper, a novel targeted metabolomics method based on isobaric tagging reagent iTRAQ®-LC-MS/MS was developed for the exploring of 42 common amino acids and related amines alterations in rats treated with hydrazine, aiming to discover the potential biomarkers for early diagnosis of DILI. Forty-two amino acids and related amines were covered in this new method. Through derivatization by iTRAQ reagent, all derivatized amino acids and related amines can be separated and quantified in 16 min with excellent peak shape and good separation efficiency. Fragments related to reporter group (m/z 113 or 121) of iTRAQ reagents could be generated from all derivatized amino acids and related amines under general multiple reaction monitoring (MRM) parameters. Isotope dilution method was established for the quantification of amino acid, which significantly reduced the interference of matrix effect on quantitative accuracy. Using this iTRAQ®-LC-MS/MS method, the changes of amino acid metabolism were comprehensively investigated in rat serum and urine samples after DILI modeling by hydrazine. More significant changes of amino acids were observed in serum and urine samples with fold changes ranging from 0.5 to 193.7. Six significantly increased amino acids in serum, including L-citrulline, L-α-amino-adipic acid, L-tyrosine, L-glutamic acid, glycine and L-lysine, and ten amino acids and related amines in urine including L-citrulline, L-α-amino-adipic acid, L-tyrosine, taurine, β-alanine, ethanolamine, argininosuccinic acid, D,L-β-amino-isobutyric acid, γ-amino-n-butyric acid and L-glutamine, 3 of which were detected in both serum and urine. Except for L-lysine all these significantly increased amino acids and related amines possessed 92.5% to 100% specificity and sensitivity calculated at best cut-off points of their ROC curves in distinguishing control and DILI model group.