Higashidate S, Hibi K, Senda M, Kanda S, Imai K
JASCO, Japan Spectroscopic Co., Ltd., Tokyo.
J Chromatogr. 1990 Aug 31;515:577-84. doi: 10.1016/s0021-9673(01)89357-2.
3 alpha- or 3 beta-hydroxysteroids, such as bile acids (free and glycine and taurine conjugates), 3 beta-hydoxy-5-cholenic acid, pregnanediol, 5-pregnene-3 beta, 20 beta-diol and 5-pregnene-3-beta,20 alpha-diol, were converted to 3-oxosteroids by enzymatic reaction using immobilized hydroxysteroid dehydrogenase, derivatized with dansylhydrazine to the corresponding dansyl hydrazones and purified by gel permeation chromatography. The dansyl hydrazones were chromatographed on a C18 column with a tetrahydrofuran-containing eluent and detected at the level of a few femtomoles by a peroxyoxalate chemiluminescence post-column reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as a chemilumigenic reagent. The dansyl hydrazones of chenodeoxycholic acid and deoxycholic acid (free and glycine and taurine conjugates) in particular, which coeluted under the chromatographic conditions above, were separated using an eluent including acetonitrile and 2,6-di-O-methyl-beta-cyclodextrin and detected in the same way.
3α-或3β-羟基类固醇，如胆汁酸（游离型以及与甘氨酸和牛磺酸的共轭物）、3β-羟基-5-胆烯酸、孕二醇、5-孕烯-3β,20β-二醇和5-孕烯-3β,20α-二醇，通过使用固定化羟基类固醇脱氢酶的酶促反应转化为3-氧代类固醇，用丹磺酰肼衍生化为相应的丹磺酰腙，并通过凝胶渗透色谱法纯化。丹磺酰腙在含有四氢呋喃的洗脱剂的C18柱上进行色谱分离，并使用双[4-硝基-2-(3,6,9-三氧杂癸氧基羰基)苯基]草酸酯作为化学发光试剂，通过柱后过氧草酸酯化学发光反应在飞摩尔水平进行检测。特别是鹅去氧胆酸和脱氧胆酸（游离型以及与甘氨酸和牛磺酸的共轭物）的丹磺酰腙，在上述色谱条件下会同时洗脱，使用包含乙腈和2,6-二-O-甲基-β-环糊精的洗脱剂进行分离，并以相同方式进行检测。
