Analysis of Inborm Errors by High-Performance Liquid Chromatography

在这个项目中，我们开发了与先天性缺陷、先天性甲状腺功能减退症和先天性肾上腺增生症等生物物质相关的高灵敏高效液相色谱方法。研究结果如下：1.建立了以固定化3-羟基类固醇脱氢酶柱为柱后酶反应器测定先天性肾上腺增生症患者尿液中糖皮质激素及其代谢物的荧光高效液相色谱方法。在检测器的最高灵敏度下，以2.5ug/dl的浓度测量每种类固醇。平均回收率为91.5%~108.2%，重现性为0.9%~6.5%(CV%)。基于荧光素的化学发光反应，建立了测定胆汁酸类皮质类固醇及其代谢物和苯乙酸酯的化学发光高效液相色谱分析方法。类固醇类化合物的检出限约为50pmol-1nmol。变异系数为4.0~9.7%。建立了以N-(4-氨基丁基)-N-乙基异硫醇为柱前试剂的化学发光高效液相色谱测定胆汁酸的方法。胆酸的化学发光导数为20fmol。这一数值高于使用3-羟基类固醇脱氢酶反应器的化学发光高效液相色谱法。建立了流动注射化学发光法测定NADH及其在高效液相色谱检测系统中的应用。该方法可用于胆汁酸及相关3-羟基类固醇的测定。建立了以苯基溴为柱前试剂测定血浆中游离脂肪酸的高效液相色谱方法。

In this project, we have developed highly sensitive HPLC methods for biological substances related to inborn errors; congenital hypothyroidism and congenital adrenal hyperplasia etc.. The results are summarized as follows :1. Fluorescence HPLC method using an immobilized 3 -hydroxysteroid dehydrogenase column as a post-column enzyme reactor has been developed for the determination of corticosteroids and their metabolites in the urine of subjects with congenital adrenal hyperplasia. Each steroid was measured at the 2.5 ug/dl at the highest sensitivity of the detector. The mean recoveries and reproducibilities were 91.5 - 108.2% with 0.9 - 6.5% (CV%).2. Chemiluminescence HPLC method based on the chemiluminescence reaction of lucigenin has been developed for the determination of corticosteroids and their metabolites, and phenacyl esters of bile acids. The detection limits of steroids were about 50 pmol - 1 nmol. The CV% was 4.0 - 9.7%.3. Chemiluminescence HPLC method using N-(4-aminobutyl)-N-ethylisolumionl as a pre-column reagent has been developed for the determination of bile acids. The chemiluminescent derivative of cholic acid was 20 fmol. This value was higher than that of chemiluminescence HPLC using 3 -hydroxysteroid dehydrogenase reactor.4. Chemiluminescence determination of NADH using flow injection system and its application to HPLC detection system have been developed. Bile acids and related 3 -hydroxysteroids could be assayed by this method.5. The HPLC method for the determination of free fatty acids in plasma by using phenacyl bromide as a pre-column reagent has also developed.