2D-HPLC同时测定小鼠胸腺中的酸性氨基酸异构体

Simultaneous determination of acidic amino acid enantiomers in the mice thymus by 2D-HPLC

目的研究D-酸性氨基酸的代谢组学，建立同时分离和定量体内样品中酸性氨基酸D、L体的离线二维HPLC法。方法利用硅胶基质ODS整体柱为第-维分离色谱柱，流动相为乙腈-三氟醋酸-水（9：0．05：91）；Chiralpak QD-1-AX色谱柱为第二维手性分离柱，流动相为含10mmol·L^-1柠檬酸的甲醇-乙腈（50：50），荧光法检测，离线手性2D—HPLC分析系统。结果两对对映体能同时得到良好地分离（R，〉2．50）；各异构体的定量线性范围均为0．005—5pmol（r〉0．9998），检出限和定量限分别为2、5fmol，回收率均在98％-105％，日内日间精密度的RSD均〈5％。应用该法测得SAMP1及SAMR1小鼠胸腺组织中D-天门冬氨酸的浓度分别为212．2、163．4nnmol·g^-1（n=6）。结论所用方法具有较高的选择性、灵敏度和准确性，可用于体内样品中酸性氨基酸异构体的同时测定。

OBJECTIVE To establish a highly selective and sensitive method for the simultaneous determination of acidic amino acid enantiomers for a further metaholomics study. METHODS An off - line 2D - HPLC method was established by adopting a monolithic ODS column in the first dimension with the mobile phase of MeCN - TFA - water （9 ： 0.05 ： 91 ） , and a Chiralpak QD - 1 - AX enantioselective column was selected for the second dimension with the mobile phase of 10 mmol·L^-1 citric acid in MeOH - MeCN （50 ： 50） , and the detection was carried out by fluorescence. RESULTS Aspartie acid and glutamic acid enantiomers could be sim- uhaneously separated （ R5 〉 2.50 ） , and each enantiomers had good linearity within the range of 0. 005 - 5 pmol （ r 〉 0. 9998 ）. The LOD and the LOQ value were 2 fmol and 5 fmol, respectively. The recovery were around 98% - 105% ,RSD values for intra - day and inter - day precision were less than 5% in the biological samples. Using this method the D - Asp amount in the SAMP1 and SAMR1 mice thymus were determined as 212.2 and 163.4 nnmol·g^-1, respectively. CONCLUSION The established method is highly selective, sensitive and accurate, and can be used for the simultaneous determination of acidic amino acid enantiomers in biological samples.