Capp M W, Simonian M H
Anal Biochem. 1985 Jun;147(2):374-81. doi: 10.1016/0003-2697(85)90285-4.
The major adrenal steroids were separated by multistep gradient elution with a reversed-phase high-performance liquid chromatography system, employing water and 1-propanol as solvents. With this solvent system, a wide range of 21 5-ene-3 beta-ol and 4-ene-3-one steroids can be resolved in a single chromatogram, which was not possible with previously published gradient solvent systems. In particular, intermediate steroids of the biosynthetic pathway, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone, were separated with baseline or sufficient resolution to allow accurate quantitation. Using the 1-propanol-water gradient, the separations of 5-ene and 4-ene steroids were compared on different octadecylsilyl packings. Optimum resolution was obtained with a fully covered, spherical particle. The 1-propanol-water gradient was compared to a previously published methanol-water gradient in the analysis of steroidogenesis by adrenocortical cell cultures. HPLC analysis of the steroid production was quantitatively the same with both gradient solvent systems. However, qualitatively, the methanol-water gradient system did not resolve the above-mentioned intermediate steroids.
采用反相高效液相色谱系统，以水和1 - 丙醇为溶剂，通过多步梯度洗脱分离主要肾上腺类固醇。使用该溶剂系统，一系列21 5 - 烯 - 3β - 醇和4 - 烯 - 3 - 酮类固醇可在一次色谱分析中得到分离，这是之前发表的梯度溶剂系统无法实现的。特别是生物合成途径中的中间类固醇，17α - 羟孕酮、17α - 羟孕烯醇酮和脱氢表雄酮，以基线分离或足够的分辨率分离，从而实现准确的定量分析。使用1 - 丙醇 - 水梯度，在不同的十八烷基硅烷填料上比较了5 - 烯和4 - 烯类固醇的分离情况。使用完全覆盖的球形颗粒可获得最佳分辨率。在肾上腺皮质细胞培养物的类固醇生成分析中，将1 - 丙醇 - 水梯度与之前发表的甲醇 - 水梯度进行了比较。两种梯度溶剂系统对类固醇生成的HPLC分析在定量上是相同的。然而，在定性方面，甲醇 - 水梯度系统无法分离上述中间类固醇。
