Abstract
Amino acids (AA) play a crucial role in the metabolic process of animals, plants and microbial cells, which are useful for the diagnosis, follow-up and prognostics of liver disorders affecting AA metabolism. A rapid, simple and sensitive analytical method is in urgent need to investigate the intact metabolic profile and to simultaneously determine individual AA in biological samples. Here, a hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry (MS/MS) analytical method was developed and validated for simultaneous quantification of 15 AA in rat serum using isotope stable-labeled phenylalanine and alanine as internal standards. The 15 AA without derivatization were separated on a hydrophilic interaction silica column (TSK-GEL AMIDE-80), the total analytical time was within 8 min, and the concentrations of the 15 AA were determined using a multiple reaction monitoring (MRM) mode. Limits of detection (LOD) ranged from 0.01 to 0.05 μg/ml, and the calibration curve was linear in the range of 0.05–10 μg/ml (r > 0.99). The HILIC-MS method was employed to the analysis of AA in serum samples obtained from N-acetyl-p-amino-phenol (APAP)- and chloropromazine hydrochloride (CH)- induced liver injured rats. The concentrations of each AA ranged from the low quantification value up to 10 μg/ml. Based on metabolic profile of AA, multivariate statistics using principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) could differentiate two distinct groups corresponding to APAP-induced and CH-induced rats. This novel metabolic profile study of AA based on the HILIC-MS analysis and chemometric analysis provided not only an accurate quantitative assay of the serum concentrations of biomarkers, but also a promising methodology for evaluation of chemical-induced hepatotoxicity in reflecting AA metabolic pathway.
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