Having set up methods for gas chromatographic separation and mass spectrometric detection of free steroid MO-TMS derivatives, experiments were performed to determine whether the sulphuric, glucuronic and fatty acid conjugated steroids could be directly derivatised by MO and TMSI for GC-EIMS analysis. It was an aim in this study to develop a method for analysis of steroids both in their free and sulphate conjugated form and thus the sulphate esters that are too polar for gas chromatography need to be converted to non polar derivatives. Derivatisation of fatty acid esters and glucuronides of steroids was also important in order to estimate interference o f those compounds from brain extracts with the analysis of free steroids and sulphate esters by GC-MS, especially if the bound groups were removed by the derivatisation reaction. Finally, derivatisation of fatty esters and glucuronides would allow their monitoring by GC-MS during development of brain extraction and fractionation procedures.
Derivatisation o f sulphate esters
DHEAS and PREGS together with the same amount of tetracosane were derivatised as described in Chapter 2. Analysis by GC-MS revealed peaks eluting at the same time as
the respective free steroids with the same mass spectra. However, the yield of MO-TMS- PREG or -DHEA was low from their respective sulphates (see Table 3-2) and so further procedures were investigated for cleavage of the sulphate esters before derivatisation. A microsolvolysis method in a single step with MO-TMSI derivatisation has been described for sulphate esters of oestrogens [ 169] and this was used to determine whether cleavage o f the sulphate esters could be achieved. DHEAS, PREGS and tetracosane (10 |ig each) were microsolvolysed and derivatised as described in 2.4.1. The response ratios of the MO-TMS-derivatives to tetracosane in GC-MS were compared to those observed after direct derivatisation by MO-TMSI and other derivatisation methods which are described below and the results are shown in Table 3-2. A considerable increase in yield compared to the direct derivatisation methods was observed with microsolvolysis followed by derivatisation with MO-TMSI. By contrast, microsolvolysis followed by TBDMS- derivatisation was not successful. These relative yields between direct derivatisation of PREGS and DHEAS by MO-TMSI with and without prior microsolvolysis are comparable to those reported for oestrogens [169].
It is assumed that the microsolvolysis works similar to traditional solvolysis methods. Due to the small volume of solvent, neutralisation of the sample can be done directly with the derivatisation reagent solution. No further purification prior to derivatisation is necessary. The partial evaporation step before reaction completion is assumed to be necessary to remove ethyl acetate to a great part, which would otherwise inhibit reaction completion. TMSI has been shown to replace the S04^'-group of the 3-0H-A5-steroids PREGS and DHEAS under the above used reaction conditions. However, this transestérification is only partial as the results of the microsolvolysis experiments demonstrated. Solvolysis including the findings here is further investigated in Chapter 4 (4.2.2.4) in the context of previous sample treatments (solid phase extraction, etc.).
Derivatisation o f steroid glucuronides
Replacement of steroid glucuronide functions by TMSI has been described [25]. This could cause potential interferences in analyses of steroid tissue extracts with other
fractions. The MO-TMSI method used here was thus applied to standard PREG glucuronide. Analysis of the reaction products by GC-MS could detect no steroid derivative from injection of 1 p.g equivalent steroid. It was estimated that with this method approximately 0.75 ng of the equivalent free steroid would have been detected (scan mode).
It was then investigated whether derivatisation with the glucuronide ether intact would be more successful. The acid moiety can be guarded by méthylation [25]. This method was adapted here (see 2.4.1). After méthylation of the acid moiety with boron trifluoride- methanol, MO-TMS derivatisation yielded a compound with a double peak in the chromatogram (RIs -3190 and 3245, scan method 2, column CPSil 5 CB). The gas
chromatogram, however, showed several other peaks of which the identity could not be clarified and the signal of the putative derivative was comparatively low. These facts indicate a low yield from the reaction and formation of reaction artefacts.
Derivatisation o f fatty acid ester steroid conjugates
MO-PREG acetate yielded a single peak (RI 2854 compared to 2834 o f the free steroid, scan method 2, column CPSil 5 CB) with high response (detectability 1.8 ng in scan mode), thus indicating high reaction efficiency. Its mass spectrum could be clearly assigned to the proposed fragmentation pattern. Structural fragments, e.g. 1 0 0 m/z
indicated derivatisation by MO at C-20, however not trimethylsilylation at C-3. The acetylated carbonyl function according to this fragmentation did not react with MO. On the contrary, PREG stearate could not be detected as MO- or TMS-derivative by GC- MS (detection limit 0.75 ng of free steroid equivalent in scan mode). The GC separation was extended 1 0 minutes beyond the normal time program isothermally.
If the derivatisation reaction had worked in a similar manner to PREG acetate, a possible explanation for lack of detection of this steroid is its retention by Lipidex 5000® in the routine cleanup procedure after MO-TMS derivatisation. The intact steroid ester is both a significantly larger molecule and more hydrophobic one than the corresponding free steroid. Size exclusion and polarity are the underlying mechanisms of chromatography by Lipidex. AJternatively it could be that the applied GC conditions (scan method 2 extended by 10 minutes) were not suitable for detection of this derivative. Larger molecules o f similar chemical structure are expected to have longer elution times/higher elution temperatures. Even after the above GC run extension the steroid derivative might
not have progressed to the detector. However, it can be concluded that steroid long chain fatty acid esters do not interfere with analysis of free steroids or steroid sulphates from brain by GC-MS.
3.2.5 Development of two ion selected ion monitoring methods for