Schatzman G L, Laughlin M E, Blohm T R
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Anal Biochem. 1988 Nov 15;175(1):219-26. doi: 10.1016/0003-2697(88)90381-8.
A normal phase HPLC system has been developed which is applicable to all of the steroid separations involved in the assay of steroid 17 alpha-hydroxylase (EC 1.14.99.9) and C17-20 lyase activities, in both the delta 4 (progesterone) and delta 5 (pregnenolone) pathways. A hexane-tetrahydrofuran (THF) gradient system is used with silica stationary phase and a flow cell radioactivity detector having a high efficiency for tritium. Folch extraction produces uniform extraction of substrates and products from the microsomal incubates, and this uniformity is maintained through HPLC separation and measurement. The hexane-THF mobile phase is convenient for product isolation and substrate purification and should be adaptable to other steroid separations. The system is especially useful for steroid enzyme assays utilizing radiolabeled substrates, since internal standards are not required for measuring recoveries of substrates and products.
已开发出一种正相高效液相色谱系统，该系统适用于甾体17α-羟化酶（EC 1.14.99.9）和C17-20裂解酶活性测定中涉及的所有甾体分离，涵盖Δ4（孕酮）和Δ5（孕烯醇酮）途径。使用己烷 - 四氢呋喃（THF）梯度系统，搭配硅胶固定相和对氚具有高效率的流通池放射性检测器。Folch萃取可从微粒体孵育物中均匀萃取底物和产物，并且这种均匀性在高效液相色谱分离和测量过程中得以保持。己烷 - THF流动相便于产物分离和底物纯化，并且应适用于其他甾体分离。该系统对于利用放射性标记底物的甾体酶测定特别有用，因为测量底物和产物的回收率时无需内标。
