Simultaneous determination of hepatoma-associated tyrosine and valine amino acid enantiomers by reversed-phase high performance liquid chromatography

Abstract:

Objective：To establish reversed-phase high performance liquid chromatography-fluorescence detection（RP-HPLC-FLD） method，to determine hepatoma-associated tyrosine and valine amino acid enantiomers（L-tyrosine（L-Tyr），D-tyrosine（D-Tyr），L-valine（L-Val） and D-valine（D-Val）），to compare changes in tyrosine and valine amino acid enantiomers concentrations in plasma between hepatocellular carcinoma（HCC） patients and healthy controls and to investigate their possible relationship. Methods：O-ph－thaldialdehyde（OPA） and N-acetyl-L-cysteine（NAC） were served as the pre-column derivatization reagents. Separation was carried out on a Phenomenex Gemini C18 column（250 mm×4.6 mm，5 μm） with a programmed gradient elution. The mobile phase was con－sisted of 20.0 mmol/L sodium dihydrogen phosphate（pH 6.80） and acetonitrile. The eluted solution was monitored using a fluores－cence detector with excitation wavelength at 350 nm and emission wavelength at 450 nm. Results：The presented method exhibited an excellent linearity for all the analytes over their respective concentration ranges. The recoveries ranged from 90.74% to 106.80%. The detection limits were 6.79 pg/ml，7.81 pg/ml，1.86 pg/ml and 1.95 pg/ml for L-Tyr，D-Tyr，L-Val and D-Val，respectively. Twenty plas－ma samples of healthy humans and 23 plasma samples of HCC patients were tested and the HCC patients demonstated higher levels of L-Tyr（P=0.011） and lower levels of L-Val（P=0.019） and D-Val（P=0.006） in plasma while no change was observed in D-Tyr（P=0.973）. Conclusion：This method is simple，accurate and suitable for routine scientific research and clinical measurement.