Selective reactions in the analysis and characterization of steroids by gas chromatography-mass spectrometry.
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.
D001897:Boronic Acids; D002789:Cholesterol Oxidase; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D007202:Indicators and Reagents; D013256:Steroids / Q000032:analysis*; D013329:Structure-Activity Relationship; D014297:Trimethylsilyl Compounds
D001897:Boronic Acids; D007202:Indicators and Reagents; D013256:Steroids; D014297:Trimethylsilyl Compounds; D002789:Cholesterol Oxidase