柱前手性衍生化-HPLC法测定人血浆和尿液中D-谷氨酸和D-丝氨酸的浓度  

Determination of D-glutamic Acid and D-serine Acid in Human Plasma and Urine by Pre-column Chiral Derivatization-HPLC

作　　者：李晶[1] 刘庆[2] 肖忠华[1] 李惠芝[3] 

机构地区：[1]重庆三峡医药高等专科学校药理学教研室,重庆404120 [2]新疆医科大学附属中医医院,乌鲁木齐830006 [3]重庆医科大学药学院药物分析教研室,重庆400016

出　　处：《中国药房》2012年第34期3212-3214,共3页China Pharmacy

摘　　要：目的:建立手性分离D-谷氨酸和D-丝氨酸的方法,测定人血浆和尿液中D-谷氨酸和D-丝氨酸的浓度。方法:12对极性氨基酸标准液混合后,加入衍生化试剂邻苯二甲醛、N-乙酰-L-半胱氨酸衍生化后,采用高效液相色谱法进样测定。色谱柱为大连依利特HypersilC18,流动相为甲醇-50mmol·L-1醋酸铵缓冲液(pH6.0),梯度洗脱,流速为1.0mL·min-1,柱温为室温,进样量为20μL,荧光检测波长为350nm(激发波长)、450nm(发射波长)。结果:D-谷氨酸、D-丝氨酸检测浓度分别在0.5～20、0.5～80nmol·L-1范围内线性关系良好,平均方法回收率分别为96.4%～103.6%、97.1%～100.5%,日内、日间RSD均<8%。测得正常人血浆中D-谷氨酸和D-丝氨酸平均含量分别为(2.43±0.37)、(1.07±0.11)nmol·mL-1,尿样中未能检测到2种D型氨基酸。结论:本研究建立的手性分离测定D-谷氨酸和D-丝氨酸的分析方法可行,可用于测定人血浆和尿液中二者的浓度。OBJECTIVE： To establish a method for chiral separation of D-glutamic acid （D-Glu） and D-serine acid （D-Ser）, and to determine the concentrations of D-GIu and D-Ser in human plasma and urine. METHODS： 12 pairs of polar amino acids were mixed and derivatized by orthophthalaldehyde and N-phenacetin-L-cysteine. HPLC determination was performed on Hypersil C18 column with mobile phase consisted of methanol-50 mmol.L^-1 ammonium acetate buffer solution （pH 6.0, gradient elution） at the flow rate of 1.0 mL. min^-1. The column temperature was room temperature and injection volume was 20μL. The fluorescence wavelength were set at 350 um （λex） and 450 nm （λem）. RESULTS： The linear ranges of D-Glu and D-Ser were 0.5-20 nmol.L^-1 and 0.5-80 nmol-L^-1 with average recoveries of 96.4%-103.6% and 97.1%-100.5%. The RSDs of intra-day and inter-day were lower than 8%. The contents of D-GIu and D-Ser were （2.43 ± 0.37） nmol.mL^-1 and （1.07 ± 0.11）nmol-mL^-1 in human plasma, and they weren＇ t found in urine. CONCLUSION： Established method is feasible and suitable for the determination of D-Glu and D-Ser concentration in human plasma and urine.

关 键 词：高效液相色谱法 柱前衍生化 手性分离 荧光检测器 D-谷氨酸 D-丝氨酸 

分 类 号：R978.1[医药卫生—药品] R969.1[医药卫生—药学]