The Application of Combined Gas Chromatography-Mass Spectrometry to Compounds of Biological Interest: Steroid Analysis by the Use of Glass Open Tubular Gas Chromatographic Columns

Abstract

Steroidal compounds are virtually ubiquitous in nature and frequently occur in complex mixtures as constituents of closely similar structure. Packed column gas chromatography-mass spectrometry (GC-MS) is an analytical technique of unique facility for the simultaneous separation and characterisation of complex mixtures of organic compounds. This thesis examines the additional facility provided by glass open tubular gas chromatographic columns of higher efficiency in the analysis by GC and GC-MS of complex mixtures of steroidal compounds derived from a number of natural sources. A method for the preparation of stable, efficient and reproducible glass open tubular gas chromatographic columns is described. The construction of chromatographic systems adapted to their somewhat more critical requirements is outlined. Particular attention is paid to the requirements for the interface of these columns to an LKB 9000 combined gas chromatograph-mass spectrometer. The performance of this system with emphasis on the nature of the advantage obtained over conventional packed columns is demonstrated by several model separations of mixtures of closely related standard steroidal compounds. Mixtures of sterols derived from yeast and marine sources frequently consist in mixtures of components differing in the degree and position of alkylation and unsaturation in the basic cholestanol structure. Correlation of gas chromatographic retention, on glass open tubular columns of OV-1 stationary phase, with sterol structure is described. Complementary data available in the literature are integrated into a scheme for the rationalisation of increments of Kovats retention index associated with particular alterations in sterol structure. This system and mass spectral correlations obtained by glass open tubular GC-MS is applied to the analysis of sterol mixtures derived from five species of marine invertebrate and two mutant strains of the yeast Candida albicans. Two other applications to sterol analysis are also described. A significant advantage is demonstrated over GC and GC-MS methods heretofore employed. Mixtures of hydroxy and ketosteroids may be derivatised as the alkyloxime-trimethylsilyl ether derivatives for GC and GC-MS analysis. The occurrence of syn- and anti-isomers in the alkyloximes of various ketosteroid structures is a complicating factor in their GC analysis, in particular at higher column efficiencies. Several alkyloximes of increasing hulk of the O-alkyl substituent were examined in this respect. The methyloxime is shown to provide the least complications for open tubular GC, though the "group separations" of hydroxy and ketosteroids provided by the higher alkyloximes may provide useful correlations. The utility of this approach is demonstrated in the analysis of mixtures of standard hydroxy and ketosteroids. Preliminary results obtained on a mixture of urinary steroid hormone metabolites of the human newborn by open tubular GC-MS of the isopentyloxime-trimethylsilyl ether derivatives are reported