酪氨酸对映体的柱前手性衍生高效液相色谱-化学发光联用法研究

目的：建立酪氨酸对映体的柱前手性衍生高效液相色谱-化学发光联用新方法，并用于体内外酪氨酸对映体的高效检测。方法：利用柱前手性衍生高效液相色谱法对酪氨酸对映体进行分离，联用化学发光检测器对酪氨酸对映体进行检测。考察了色谱流动相配比、流速、柱温以及化学发光条件对酪氨酸对映体分离结果的影响。采用柱前手性衍生高效液相色谱-化学发光联用法对生物样品中的酪氨酸对映体进行分离分析。结果：最佳色谱条件下，流动相为甲醇-0.01 mol·L^-1磷酸二氢钾溶液（35：65，pH 6.0），流速为0.7 mL·min^-1，柱温为室温；最优化学发光体系为氢氧化钠、鲁米诺和高锰酸钾，其浓度分别为0.15、4.0×10^-5和4.0×10^-5 mol·L^-1。在此优化条件下，酪氨酸对映体可以达到完全分离（R_S>2.0），线性检测范围为2.5×10-7~3.2×10^-5 g·mL^-1，L-/D-酪氨酸的检测下限分别为2.96×10^-8、3.59×10^-8 g·mL^-1。结论：柱前手性衍生高效液相色谱-化学发光联用法可对酪氨酸对映体进行高选择性、高灵敏度检测。

Objective: To establish a novel HPLC-CL method with precolumn chiral derivatization for the determine of tyrosine enantiomers,and to detect chiral substances in vivo and in vitro.Methods: The tyrosine enantiomers were separated by HPLC with precolumn chiral derivatization and detected by chemiluminescence detector;the chromatographic mobile phase,flow rate,column temperature and chemiluminescence conditions were investigated;the established method was used to analyze the tyrosine enantiomers in biological samples.Results: The chromatographic mobile phase consisted of methanol and 0.01 mol·L^-1 monopotassium phosphate solution(35:65,pH 6.0),the flow rate was 0.7 mL·min^-1,and the column temperature was room temperature.The chemiluminescence system was composed of sodium hydroxide(0.15 mol·L^-1),luminol(4.0×10^-5 mol·L^-1) and potassium permanganate (4.0×10^-5 mol·L^-1).Under the optimized conditions,the tyrosine enantiomers could be completely separated(R_S>2.0) with the linear range of 2.5×10-7~3.2×10^-5 g·mL^-1,and the detection limits of L-and D-tyrosine were 2.96×10^-8 and 3.59×10^-8 g·mL^-1,respectively.Conclusion: The established method can be used to selectively and sensitively determine tyrosine enantiomers.