两种柱前衍生反相高效液相色谱法测定血液和尿液中游离氨基酸  

Determination of Free Amino Acids in Serum and Urine by Precolumn Derivatization HPLC with PITC and AQC

作　　者：马晓丽[1] 孟磊[1] 巴吐尔[1] 李琳琳[2] 李新霞[1] 孔彬[1] 勉强辉[1] 王烨[2] 毛新民[1,3] 

机构地区：[1]新疆医科大学分析测试中心,新疆乌鲁木齐830011 [2]新疆医科大学基础医学院,新疆乌鲁木齐830011 [3]新疆医科大学中医学院,新疆乌鲁木齐830011

出　　处：《氨基酸和生物资源》2012年第3期19-24,共6页Amino Acids & Biotic Resources

基　　金：国家自然科学基金(81260144);新疆地方病分子实验室开放课题(XJDX0208-2010-02)

摘　　要：建立以PITC法和AQC法为柱前衍生试剂测定血液和尿液中游离氨基酸含量的测定方法。采用Waters-e2695操作系统,色谱柱为Shim-vp,ODS(250mm×4.6mm,5μm)(日本,岛津公司),以甲醇/乙腈/水和醋酸钠溶液(pH 6.5)为流动相,梯度洗脱。分别采用紫外和荧光检测器对血液和尿液中游离氨基酸进行含量测定。结果显示,两种衍生化方法灵敏度好、分离度高,具有良好的线性范围(r>0.990 0),准确度高(平均回收率为75.1%～127.0%),进样精密度好(RSD为0.12%～3.42%)。PITC法在尿液中游离氨基酸含量测定中显示了良好的测试准确性;而AQC法测定尿液中组氨酸、苏氨酸、脯氨酸超出线性范围,需要对尿样的前处理进行深入研究。A precolumn derivatization HPLC method for the determination of free amino acids in plasma and urine with PITC and AQC was established. The determination was performed on waters e2695 system, using a Shim-vp, ODS column with mobile phases of methanoi/acetate/H20 and sodium acetate buffer（pH 6.5）, gradient elution, the content of amino acid were tested by UV and fluorescence detector, respectively by PITC and AQC. The result was sensitive and accurate. Both of the two derivatization reagents showed good linear range（r〉0. 990 0）, with an average recovery ranged from 75.1% to 127.0%. The injection RSD was within 0.12-3.42G. PITC was validated and used to the determination of urine sample which showed good quantitation. A few kinds of amino acids （His, Thr, Pro） beyond the liner range when using AQC method, which should be further studied.

关 键 词：柱前衍生 高效液相色谱法 异硫氢酸苯酯 6-氨基喹啉-N-羟基琥珀酰亚胺基氨基甲酸酯(AQC) 血液 尿液 游离氨基酸 

分 类 号：R917[医药卫生—药物分析学]