Colin Frank Poole
An investigation into the analysis of ecdysones and other steroid hormones
Poole, Colin Frank
As an aid to the analysis of ecdysones (arthropod moulting hormones), an investigation has been carried out to find an efficient and sensitive method for determining them by chromatography.
A series of fluorocarbonsilanes were synthesized and tested as electron capturing derivatives for gas chromatography. Flophemesyl* (*pentafluorophenyldimethylsilyl) derivatives of mammalian steroids were volatile and sensitive to detection. The derivatives produced characteristic mass spectra with a greater proportion of the ion current carried by hydrocarbon fragments than with other siloxy derivatives making flophemesyl derivatives more suitable for structural determinations. A series of flophemesyl reagents were developed for selectively protecting hydroxyl groups in different steric environments. New methods of synthesis of some of these reagents and analytical techniques for their identification were established.
In their mass spectra, unusual rearrangements were found to occur between the fluorine atoms of the pentafluorophenyl ring and the methyl groups bonded to silicon, giving hydrofluorocarbon tropylium-type ions.
Flophemesyl derivatives, although useful for mammalian steroids did not give volatile derivatives of ecdysones.
The introduction of the pentafluorophenyl ring into steroids as pentafluorophenylhydrazones or pentafluorophenylboronic esters was attempted. The hydrazones, as ketone derivatives, had poor GC characteristics and limited stability when exposed to light or the atmosphere. The pentafluorophenylboronic esters were susceptible to disproportionationin the presence of traces of water or other strong nucleophiles.
Boron can be selectively detected with a nitrogen thermionic detector. Boronic esters of model steroids were detected at lower levels than with the FID, but the estimated sensitivity for ecdysones was insufficient for trace level analysis.
As ecdysone TMS ethers fragment to produce mass spectra containing a few ions of relatively high intensity, the possibility of using the mass spectrometer as a gas chromatographic detector was investigated. The ecdysone derivatives could only be chromatographed at high temperatures as low carrier gas flow rates had to be used to meet the vacuum requirements of the mass spectrometer. An excessive column background made quantification difficult at trace levels. Optimum GC-MS conditions for steroid analysis were established.
The model steroids 28,30-dihydroxy-5a-cholestane and 28,3$,lUa-tri- hydroxy-50-cholest-7-en-6-one were prepared by published procedures; 5a-cholest-7-en-6-one and 14a-hydroxy-5o-cholest-7~en-6-one were synthesised for the first time in good overall yield.
In an investigation of the formation of ecdysone THS ethers and the formation of heptafluorobutyrate esters by an exchange reaction with the TMS ethers, it was discovered that the ecdysone nucleus contains the features necessary for electron capture without the need for the formation of halogenated derivatives. The electrophore was identified as the unsaturated ketone, the C-14 oxy substituent with a smaller contribution from the more remote 28,30-oxy substituents. The rate of formation of the TMS ethers of ecdysterone hydroxyl groups with trimethylsilylimidazole was found to be 2,3,22,25>20>>1*U The degree of TMS ether formation in ecdysone and ecdysterone was confirmed by mass spectrometry of the derivatives and also by selective silylation of model steroids. Ecdysones in biological samples were determined as their TMS ethers after a preliminary extraction, solvent partition and TLC separation from impurities. The method has been applied to determine the daily changes in ecdysone content in the desert locust, Schistocerca gregaria.
A brief survey of high pressure liquid chromatography illustrated the potential of this technique for the detection and separation of ecdysones.
|Publication Date||Sep 1, 1975|