Shackleton C H, Mattox V R, Honour J W
J Steroid Biochem. 1983 Jul;19(1A):209-17.
The analysis of intact steroid conjugates by two different methods is described. One method employed secondary ion mass spectrometry (SIMS) using a Cs+ beam for ionisation, although comparable data were obtained by fast atom bombardment (FAB) using a Xe0 beam. In both of these mass spectrometric techniques the samples were analysed in a liquid matrix (glycerol). Positive and negative ion spectra have been obtained, the latter being most useful for steroid sulphate and glucuronide analysis. The negative ion spectrum of each steroid is dominated by a pseudomolecular ion at m/z [M - H]- (M of free acid) and a lack of marked fragmentation. Mixtures of steroids can be resolved in a single spectrum, providing the individual steroids differ in mass. The second method was gas chromatography. The carboxylic acid moieties of the steroid glucuronides were derivatised with diazomethane and the remaining functional groups in the steroids were thermally protected by methyloxime formation (for carbonyls) and trimethylsilylation (for all steroidal and glucuronic acid hydroxyls). Satisfactory analysis of steroid glucuronides was achieved through the use of glass or fused silica columns stable at high temperature (330 degrees C). Conveniently, trimethylsilylation resulted in exchange of the sulphate in 3 beta-hydroxy-5-ene steroid sulphates for a trimethylsilyl group so these could effectively be analysed as "free" steroids.
本文描述了用两种不同方法对完整甾体缀合物进行分析的过程。一种方法采用二次离子质谱法（SIMS），使用Cs⁺束进行电离，不过使用Xe⁰束的快原子轰击（FAB）也能获得类似的数据。在这两种质谱技术中，样品均在液体基质（甘油）中进行分析。已获得正离子和负离子光谱，其中负离子光谱对甾体硫酸盐和葡糖醛酸苷的分析最为有用。每种甾体的负离子光谱以m/z [M - H]⁻处的准分子离子（游离酸的M）为主，且碎片较少。甾体混合物可在单张光谱中得到分离，前提是各个甾体的质量不同。第二种方法是气相色谱法。甾体葡糖醛酸苷的羧酸部分用重氮甲烷进行衍生化，甾体中的其余官能团则通过形成甲基肟（针对羰基）和三甲基硅烷基化（针对所有甾体和葡糖醛酸的羟基）进行热保护。通过使用在高温（330℃）下稳定的玻璃柱或熔融石英柱，实现了对甾体葡糖醛酸苷的满意分析。方便的是，三甲基硅烷基化导致3β-羟基-5-烯甾体硫酸盐中的硫酸盐被三甲基硅烷基取代，因此这些化合物可作为“游离”甾体进行有效分析。
