反相高效液相色谱法同时测定肝癌相关酪氨酸、缬氨酸对映体

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

摘要:

目的：建立柱前衍生反向高效液相色谱荧光检测法同时对肝癌相关L-酪氨酸（L-tyrosine，L-Tyr）、D-酪氨酸（D-tyro－sine，D-Tyr）、L-缬氨酸（L-valine，L-Val）、D-缬氨酸（D-valine，D-Val）进行拆分测定。比较正常人与原发性肝细胞癌（hepato－cellular carcinoma，HCC）（以下简称原发性肝癌）患者血浆中酪氨酸与缬氨酸对映体含量的变化，考察它们与原发性肝癌的关系。方法：柱前衍生化试剂：邻苯二甲醛（O-phthaldialdehyde，OPA）和N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine，NAC）；色谱柱：菲罗门Gemini C18柱（250 mm×4.6 mm，5 μm）；流动相：20.0 mmol/L 磷酸二氢钠（pH 6.8）-乙腈为流动相的梯度洗脱；荧光检测波长设定：激发波长（λex）为350 nm，发射波长（λem）为450 nm；流速1 ml/min；柱温30 ℃；进样量10 μl。结果：４种物质的回收率在90.74%~106.80%之间，线性关系良好，检出限分别为6.79 pg/ml（L-Tyr），7.81 pg/ml（D-Tyr），1.86 pg/ml（L-Val），1.95 pg/ml（D-Val）。利用该方法，对20名健康志愿者和23名HCC患者的血浆进行测定，统计分析结果表明，L-Tyr明显升高（P=0.011），L-Val（P=0.019）、D-Val（P=0.006）显著性降低，D-Tyr（P=0.973）无明显变化。结论：该方法准确、灵敏、重复性好，适用于实验室研究与临床检测。

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.