Kessler M J
Steroids. 1982 Jan;39(1):21-32. doi: 10.1016/0039-128x(82)90122-2.
Rapid separation of gonadal steroids in the progesterone (delta 4) and pregnenolone pathway (delta 5) has been accomplished by the use of high performance liquid chromatography (HPLC). Two HPLC systems are utilized. THe first requires the use of two separate radial compression columns (C-18 and C-8), with steroids being eluted with a methanol-water gradient. The second employs a stainless steel C-18 (reversed phase) column with a 12% octadecylsilane coating. The latter system separates seven of the eight steroids in the delta 4 and delta 5 pathways in thirty-five minutes. For the quantitation of steroids directly, integration of the peak areas, using 254 nm absorption for the delta 4 pathway steroids (5 ng minimum limit), and 210 nm absorption for the delta 5 pathway steroids (25 ng minimum limit) is used. For the quantitation of radiolabeled metabolites resulting from incubation of gonadal tissue with radiolabeled steroid precursors, either one of two methods is used: (1) the eluent can be recovered from the HPLC using a fraction collector, and counted in liquid scintillation counter or (2) the entire eluent (or a portion of it) can be counted immediately by directing the flow through a radioactivity detector.
通过高效液相色谱法（HPLC）已实现孕酮（δ4）和孕烯醇酮途径（δ5）中性腺甾体的快速分离。使用了两种HPLC系统。第一种需要使用两个单独的径向压缩柱（C-18和C-8），甾体用甲醇 - 水梯度洗脱。第二种采用涂有12%十八烷基硅烷的不锈钢C-18（反相）柱。后一种系统在35分钟内分离出δ4和δ5途径中的八种甾体中的七种。为了直接定量甾体，使用峰面积积分，对于δ4途径甾体使用254nm吸收（最低检测限5ng），对于δ5途径甾体使用210nm吸收（最低检测限25ng）。为了定量性腺组织与放射性标记的甾体前体孵育产生的放射性标记代谢物，可使用以下两种方法之一：（1）可以使用馏分收集器从HPLC中回收洗脱液，并在液体闪烁计数器中计数，或者（2）通过使流动通过放射性检测器可以立即对整个洗脱液（或其一部分）进行计数。
