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Metabolic profiling of fifteen amino acids in serum of chemical-induced liver injured rats by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

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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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Abbreviations

AA:

Amino acids

HILIC:

Hydrophilic interaction liquid chromatography

MS/MS:

Tandem mass spectrometry

MRM:

Multiple reaction monitoring

APAP:

N-acetyl-p-amino-phenol

CH:

Chloropromazine hydrochloride

IEX-LC:

Ion exchange chromatography

HPLC–MS:

High performance liquid chromatography-mass spectrometry

GC–MS:

Gas chromatography-mass spectrometry

PPT:

Protein precipitation

PCA:

Principal component analysis

PLS-DA:

Partial least squares discriminate analysis

VIP:

Variable importance in the projection

IS:

Internal standard

Phe:

Phenylalanine

Trp:

Tryptophan

Tyr:

Tyrosine

Leu:

Leucine

Ile:

Isoleucine

Val:

Valine

Met:

Methionine

Pro:

Proline

His:

Histidine

Asn:

Asparagine

Arg:

Arginine

Thr:

Threonine

Ala:

Alanine

Gln:

Glutamine

Lys:

Lysine

CMC-Na:

Sodium carboxymethyl cellulose

i.g.:

Oral administration

i.p.:

Intra-peritoneal

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

ALP:

Alkaline phosphatase

ALB:

Albumin

TBIL:

Total bilirubin

QC:

Quality control samples

LOQ:

Low limit of quantification

LOD:

Limit of detection

RSD:

Relative standard deviation

BCAA:

Branched chain amino acids

AAA:

Aromatic amino acids

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Acknowledgments

The financial support of the Natural Science Foundations of China (30701104 and 81073027), National S&T Major Special Projects (2009ZX09502-013, 2009ZX09502-021 and 2009ZX09502-024), Shanghai Rising-Star Program (09QA1405500) and Scientific Innovation Project from Shanghai Municipal Education Commission (09YZ125) is gratefully acknowledged. The authors are in deep debt to Prof. Karl. W. K. Tsim for his great help in English.

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Authors and Affiliations

  1. The Ministry of Education (MOE), Key Laboratory for Standardization of Chinese Medicines, Institute of Traditional Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 201210, Shanghai, China

    Ying Xu, Li Yang, Fan Yang, Zhengtao Wang & Zhibi Hu

  2. The State Administration of TCM (SATCM), Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Traditional Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 201210, Shanghai, China

    Ying Xu, Li Yang, Fan Yang, Zhengtao Wang & Zhibi Hu

  3. Department of Pharmacogonosy, China Pharmaceutical University, 210038, Nanjing, China

    Yinhua Xiong & Zhengtao Wang

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  1. Ying Xu
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  2. Li Yang
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  3. Fan Yang
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  4. Yinhua Xiong
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  5. Zhengtao Wang
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  6. Zhibi Hu
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Correspondence to Li Yang or Zhengtao Wang.

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Xu, Y., Yang, L., Yang, F. et al. Metabolic profiling of fifteen amino acids in serum of chemical-induced liver injured rats by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. Metabolomics 8, 475–483 (2012). https://doi.org/10.1007/s11306-011-0333-0

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  • DOI: https://doi.org/10.1007/s11306-011-0333-0

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