Steroids are usually derivatised to protect thermally labile compounds at the elevated temperatures in GC and may also avoid tailing of polar compounds or increase the response o f a compound in the detection. Methods most commonly used for derivatisation of compounds for GC are silylation, alkylation, oxime formation, and acylation. The most versatile silylation reagent for steroids is trimethylsilylimidazole (TMSI). TMSI reacts with hydroxyl groups to form ethers and is usually used in combination with methoxyamine hydrochloride (MO), which reacts with keto-functions to give oximes [35]. TMSI on its own can also be used to react with keto-functions, forming enols. MO derivatisation has the disadvantage o f formation o f syn/anti - isomers that can separate in GC. Hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS) are milder silyl donors and are only used for few steroids. Higher alkyl- dimethylsilyl (e.g. ré?r/-butyldimethylsilyl (TBDMS)-donors) derivatising reagents have been investigated for use in steroid analysis. They react with hydroxyl groups to give TBDMS ethers, ketone functions react after énolisation [154,155], These TBDMS- derivatives are less susceptible to fragmentation in MS and give few highly abundant ions. This makes them useful for quantitation in high sensitivity analyses. However there is a loss in structural information and identification is less specific. Sensitivities achieved were reported in the range of 1 (testosterone) and 5 pg (progesterone) in analyses of TBDMS-derivatives with GC-EIMS [5].
Acylation with fluoro-organic acids has also been employed for high sensitivity steroid analysis (e.g. trifluoroacetic (TFAA), pentafluoropropionic (PFPA) and heptafluorobutyric acid (HFBA) (used in anhydride form)). These compounds are highly electronegative and are thus especially useful for electron capture detection and negative ion chemical ionisation (NICI) MS. In BIMS, they show little fragmentation and have prominent molecular ions. They also react with keto-groups to form enols. The solvent used with these reagents plays a reaction-modifying role. Using derivatisation by HFBA in ethyl acetate, PREG, PROG, 3a, 5a- and 3a,5p-THPROG could be detected in
extracts of cerebrospinal fluid by GC-NICIMS [195]. Several oestranes, androstanes and pregnanes could be derivatised with HFBA in acetonitrile at 80°C [32]. Cortisol [191], PREG, DHEA, PROG and 3a,5a-THPROG [106] were derivatised with HFBA by using acetone as solvent at room temperature. Several corticosteroids could successfully be derivatised using benzene as solvent and heating at 60°C [118]. Sensitivities achieved were 5 pg for the pregnanes in GC-NICIMS [195], 30 pg for cortisol by GC-EIMS [191] and between 0.5-2.5 pg for the corticosteroids by GC with electron capture detection [118].
Steroid conjugate derivatisation has been extensively investigated. Steroid sulphate conjugates were found to derivatise with TMSI and HFBA by exchange with the sulphate groups [171,192]. The resulting derivatives can be analysed as their respective free steroids. Steroids are also derivatised after cleavage of the sulphate groups prior to derivatisation with chemical hydrolysis (see Introduction, Chapter 4). A microsolvolysis method in a single step with MO/TMSI derivatisation has been described for sulphate esters of oestrogens [169]. MO in pyridine was added directly after incubation of the steroids in a small volume of acidified ethyl acetate. The yields were found as high as after conventional solvolysis and derivatisation. Steroid glucuronides can be derivatised after méthylation of the acid moiety with MO at ketone functions and at the hydroxylgroups with TMSI [169]. A direct derivatisation method using TBDMS donors was also devised [185].
GC-columns
Early problems in wall coated open tubular (WCOT) capillary columns of adsorption and stationary phase contraction have been overcome through surface deactivation and the introduction of fused silica as column support material, which has a more consistent composition than previously used materials. WCOT columns have low theoretical plate heights due to uniform mass transfer path lengths and high mass transfer rates. Furthermore, the small drop in pressure allows the use o f long columns, which have higher numbers of theoretical plates. A large variety of stationary phases is available for GC. They are usually based on liquid polymers of methyl siloxane or ethylene glycol. To immobilise the phase in the column, increasing reproducibility of performance and life span, chemical bonding to the column material is now usually applied. Special low bleed columns, caused by cross-linking of the stationary phase to the support are now also
available for sensitive detectors such as MS. Such columns are used in the present study with CPSEL5 CB or equivalent stationary phases.
Samples in solvent can be applied to the GC-column in the split-, splitless- or on-column modes. In split injection, the injection volume (typically 1-2 pi) is reduced after evaporation in the heated injection chamber (typically 250-300°C) as the injector is constantly purged with a subsidiary carrier gas stream. A split-ratio of more than 100 is usually applied. The main disadvantage of this technique is the great loss of sample. In most cases this does not present a problem, except in trace analyses. A method allowing the great majority of the sample to be transferred onto capillary columns is the splitless technique devised by Grob [67]. A similar injector system as in split injection is used, but the split valve is initially kept closed. After this initial period, the injector is purged with the carrier gas stream to avoid tailing effects in the chromatogram and build up of sample residue in the injector. The technique uses a solute focusing effect that occurs as the solvent recondenses in the initial stretch of column that is held below the temperature of the sample solvent boiling point. Also for trace analysis, on-column injection can be performed, introducing the whole sample in liquid form into the column.
The most commonly used detector in capillary GC is not MS but the flame ionisation detector (FID) which has the widest range of applications. This GC method is usually used for quantitations in metabolic profiling of known urinary steroids [76]. Additional detectors for specific groups of compounds on GC are electron capture, nitrogen phosphorus and flame photometry detectors.