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The analytical characterization, by GC-MS, of individual compounds in mixtures of steroids, such as occur frequently in biological extracts, is difficult because of the close similarities in structure and properties of many components. The improved separating power of capillary (open-tubular) columns alleviates the problem, but does not solve it fully: for example, the coincidence of retention times of two different compounds may still be virtually complete. Comparative analyses on two distinctively different phases afford one valuable application of selectivity, but may not always be feasible when costly columns are required. Comparative analyses of the sample, before and after effecting its modification by well-defined reactions, are inexpensive and are particularly when selective transformations are used. The use of the microbial enzyme cholesterol oxidase as a selective oxidant for 3 beta-hydroxysteroids (chiefly limited to 4-ene, 5-ene and 5 alpha-types) is illustrated for a model mixture of androstanols related to the boar pheromone (5 alpha, 16-androsten-3 alpha-ol). Retention regularities and changes in mass spectra enhance the reliability of identifications. An exploratory application of cholesterol oxidase in the analysis of minor "polar" sterols in human serum is reported. Most of the known minor sterols are good substrates for the enzyme, and their transformation products yield distinctive GC-MS data, as exemplified for the 7 alpha- and 7 beta-hydroxycholesterols. Another convenient and versatile selective reagent is methaneboronic acid, which yields cyclic esters of suitably constituted diols. These derivatives have shorter retention times (on "non-polar" phases) than the di-TMS ethers, chiefly by virtue of their much lower molecular weights. The mass spectra of cyclic boronates generally show clear molecular ions, also fragmentations that complement the information obtainable from the di-TMS ethers. These features are illustrated for a group of diols and triols of the 5 alpha-pregnane series.

Gas chromatography-mass spectrometry (GC-MS) is a technique especially suitable for the analysis and characterization of steroids, and its power has been extensively demonstrated. The efficacy of GC-MS is limited, nevertheless, by the fact that steroid mixtures - whether of natural origin only, or augmented by synthetic analogs - often contain similar components that are poorly distinquished. The fortuitous overlap of gas chromatographic peaks from disparate compounds also impairs the definition of retention data. Controlled modification of the sample by means of selective reactions is therefore a valuable adjunct to the application of GC-MS. Two examples are discussed: (a) the enzyme cholesterol oxidase, isolated from various microorganisms, catalyzes the oxidation of many 3 beta-hydroxy-5-enes (with concomitant isomerization) to 4-en-3-ones; 3 beta-hydroxy-5 alpha-steroids are also oxidized to the corresponding 3-ones, but other steroids (3 alpha-hydroxy- or 5 beta-isomers, etc.) are unaffected. The mild conditions required (pH 7, 30 C) are advantageous for the analysis of sensitive steroids, and the retention index increments, as well as the mass spectra of the ketones, are characteristic. The enzyme accepts as substrates a wide range of 3 beta-hydroxysteroids, tolerating oxygenation in ring B and even catalyzing the oxidation of 2-oxacholesterol to the expected lactone; and (b) Steroids possessing 1,2-diol or 1,3-diol groupings include estriols, 2-hydroxyestrone, 20,22-dihydroxycholesterols, ecdysones, brassinolide and many corticosteroids. The selective formation of cyclic derivatives can provide several analytically useful features, such as convenient retention times, moderate mass increments (24 amu for a methaneboronate), distinctive mass spectra and usually abundant molecular ions. These are exemplified for 5-pregnene-3 beta, 20,21-triols and for 20,22-dihydroxycholesterol as well as its enzymic oxidation product.