Applications of Gas Chromatography - Mass Spectrometry in Steroid Chemistry (1973)

Steroids have, for many years, been recognised as an important class of compounds, because of their occurrence in almost all living systems and widespread use in pharmaceutical preparations. Many steroids, e. g. the corticosteroids, possess hormonal activity, and their analysis in biological fluids is consequently of considerable interest. In the work described in this thesis, gas-liquid chromatography (GLC) and combined gas chromatography-mass spectrometry (GC-MS) were applied to the analysis of steroids of biological significance, with particular interest centring on the corticosteroid family. As a result of the thermal lability of many of these compounds, particularly those possessing the "dihydroxyacetone" side-chain, modification of the basic structure is a prerequisite in any gas-phase analytical procedure. Of the two approaches currently employed in this area, namely oxidative cleavage of the corticosteroid side-chain, and conversion of functional groups to suitable derivatives, the latter technique was studied in some detail. The possible value of the 17alpha,21-anhydro (oxetanone) derivative in stabilising steroidal dihydroxyacetones for gas chromatography was investigated for fourteen representative examples of this type. Although these cyclic derivatives were found to possess desirable GC-MS properties, their application to analytical separations was hindered by the lack of a suitable preparative method. A number of other derivatives which have been described previously for the gas-phase separation of corticosteroids were also examined; these included trimethylsilyl (TMS) ethers, O-methyloxime trimethylsilyl (MO-TMS) ethers, acetonides, dimethylsiliconides and cyclic boronate esters. These derivatives were compared with respect to their ease of preparation, stability, retention characteristics and mass spectra. The technique of "single ion monitoring" (SIM) was applied to the quantitative detection of five representative derivatives of Reichstein's Substance S. The sensitivity with which single, characteristic fragment ions in their mass spectra could be measured varied considerably between derivatives, the limit of detection in the most favourable case (the 17alpha,21-anhydro derivative) being 400 pg. However, the derivative of choice for general use was found to be the MO-TMS ether, on account of its convenience of preparation, range of detectable concentration and applicability to all six types of corticosteroid side-chain. In view of the above findings on the suitability of oxime-TMS ethers for the characterisation of steroids by GC-MS, several O-alkylhydroxylamines were evaluated as reagents for the conversion of reactive ketosteroids to their O-alkyloxime derivatives. O-Butyloximes and O-pentyloximes proved to be useful in this respect by virtue of their structurally informative mass spectra and the convenient increments in retention time accompanying their formation. In particular, O-isopentyloxime trimethylsilyl (iPO-TMS) ethers were of value in affording gas-phase "group" separations of keto- from hydroxysteroids. This type of derivative, together with the O-benzyloxime trimethylsilyl (BO-TMS) ether, was applied to the GC-MS determination of urinary steroid profiles from newborn infants: samples from both normal and pathological cases were examined. An advantage of the iPO-TMS ethers in profile analysis was that derivatives of steroidal diketones were eluted at normal operating temperatures, whereas di-BO derivatives remain undetected due to their excessively long retention times. In the "glucuronide" fraction from a normal infant, three such compounds were detected and partially characterised. The results presented in this thesis confirm the scope and potential of GC-MS in steroid chemistry. In particular, the technique affords a highly powerful means by which complex mixtures, mixtures, such as steroid hormone metabolites in urine, may be examined. The sensitivity and selectivity of GC-MS in this area may be enhanced by single or multiple ion detection methods, and by the use of high-efficiency GLC solumns. The current development of relatively low-cost GC-MS systems could well provide the clinical chemist with an invaluable tool for both routine analysis, and metabolic research purposes.