Cravedi J P, Delous G
I.N.R.A.-Laboratoire des Xénbiotiques, Toulouse, France.
J Chromatogr. 1991 Apr 5;564(2):461-7. doi: 10.1016/0378-4347(91)80514-d.
We have recently observed in trout that 48 h after ingestion of a single dose of [14C]-17 alpha-methyltestosterone ([14C]-MT), 25% of the radioactivity was still in the carcass, corresponding to metabolites of 17-MT. These compounds have no appreciable chromophore, fluorophore or electrophore, therefore the usual detection systems was not satisfactory for their analysis. Consequently a method was developed for high-performance liquid chromatographic separation and detection of two of the major tissue metabolites of 17-MT: 5 alpha-androstane-17 alpha-methyl-3 alpha,17 beta-diol and 5 beta-androstane-17 alpha-methyl-3 alpha,17 beta-diol. A column of immobilized 3 alpha-hydroxysteroid dehydrogenase was prepared and used for detection. The NADH produced from 3-hydroxysteroids by this immobilized enzyme reactor was monitored fluorimetrically. The detection limit of this method, as obtained from the calibration curve, was at the picomole level; the limit of quantification in muscle was 1 microgram/kg, at a signal-to-noise ratio of 4.
我们最近在虹鳟鱼中观察到，摄入单剂量的[14C]-17α-甲基睾酮（[14C]-MT）48小时后，25%的放射性仍存在于鱼体中，这对应于17-MT的代谢产物。这些化合物没有明显的发色团、荧光团或电泳基团，因此常用的检测系统对其分析并不令人满意。因此，开发了一种用于高效液相色谱分离和检测17-MT的两种主要组织代谢产物的方法：5α-雄甾烷-17α-甲基-3α,17β-二醇和5β-雄甾烷-17α-甲基-3α,17β-二醇。制备了固定化3α-羟基类固醇脱氢酶柱并用于检测。通过该固定化酶反应器由3-羟基类固醇产生的NADH通过荧光法进行监测。根据校准曲线，该方法的检测限为皮摩尔水平；肌肉中的定量限为1微克/千克，信噪比为4。
