高效液相色谱——紫外、荧光双检测器同时测定人尿中犬尿氨酸和犬尿喹啉酸

目的建立高效液相色谱（HPLC）同时测定人尿中犬尿氨酸（KYN）和犬尿喹啉酸（KYNA）方法。用KYNA／KYN比值评价人体犬尿氨酸氨基转移酶（KAT）活性。方法采用HPLC测定KYN、KYNA，分析柱为安捷伦HCC18反相色谱柱；流动相为10mmol／L乙酸钠-乙酸缓冲液：7％乙腈=93：7；流速1mL／min；检测器波长：紫外365nm，荧光344nm（激发）／398nm（发射）。结果KYN和KYNA保留时间分别为6．9和11．5min。水溶液标准与标准加入工作曲线斜率差异无统计学意义（KYN：P=0．4068；KYNA：P=0．6462）。KYN和KYNA的平均回收率分别为98．74％±7．53％、95．17％±8．17％；稳定性试验的变异系数（CV）分别为3．35％和0．88％；精密度CV分别为2．73％和2．79％；最低检测限分别为0．4和0．2μmol／L。30份健康成年人尿样的KYNA／KYN比值为2．45±1．54。尿中KYNA与KYN含量呈正相关关系（r2=0．3758，P〈0．001）。结论测定方法灵敏、准确、稳定。KYNA／KYN比值评测KAT活性具有简便、实用等特点，可用于相关疾病的临床辅助诊断和基础研究。

Simultaneous determination of kynurenine and kynurenic acid in human urine by HPLC with ultraviolet and fluorescence detector

Objective To establish the high performance liquid chromatography （HPLC） method for determining kynurenine （KYN） and kynurenic acid （KYNA） in human urine simuhaneously. To evaluate the activity of human kynurenic aminotransferase （KAT） with KYNA/KYN ratio. Methods The levels of KYN and KYNA were determined by HPLC. Analytical column： Agilent HC C18 column. Mobile phase： a buffer solution of 10 mmol/L sodium acetateacetic acid ：7% acetonitrile = 93 ： 7. Flow rate： 1 mL/min. Wavelengths of detectors： ultraviolet, 365 nm; fluorescence, excitation 344 nm/emission 398 nm. Results The retention times of KYN and KYNA were 6.9 and 11.5 min respectively. There was not significant difference between the slopes of linear regression curves of aqueous and urinematched standard-addition curves （ KYN ： P = 0. 406 8 ; KYNA： P = 0. 646 2）. The average recoveries of KYN and KYNA were 98.74% ± 7.53% and 95.17% ± 8.17%. The coefficients of variation （CV） of KYN and KYNA were 3.35 % and 0.88 % for stability test,2.73 % and 2.79 % for precision test. The lowest detection limit of the method was 0.4 μmol/L for KYN and 0.2 tLmol/L for KYNA. The KYNA/KYN ratio of 30 urine samples of healthy subjects was 2.45 ±1.54. There was positive correlation between KYNA and KYN levels in urine （r2 = 0. 375 8, P 〈 0. 001 ）. Conclusions The method is sensitive, accurate and stable. Using KYNA/KYN ratio to determine the activity of KAT has the characteristics of convenience and practicability and this method could be used for the clinical diagnosis and fundamental research for some relative diseases.