Gas Chromatography - Tandem Techniques - Examples of GC/MS Spectrometry Applications > Analysis of Anabolic Steroids in Biological Materials > Page 73
The hydrolyzed conjugates were eluted from the cartridge with ether and then washed with 2 mol l-1 NaOH, dried over sodium sulfate and the solvents again removed in vacuo. The trimethyl silane derivatives were prepared by heating the fraction with 8% methoxyamine hydrochloride in pyridine at 80ūC for 30 min, the pyridine was then removed in vacuo and the residue heated with N-methyl-N-(trimethylsilyl)-trufluoroacetimide at 80 ūC for 1 hr. Excess reagent was also removed in vacuo and the residue dissolved in n-decane. The mass spectrometer employed was the Finnigan MATTSQ 70 and the separation was carried out on a BPX5 column 25 m long, 250 mm I.D. The column was operated isothermally at 150ūC for one minute, programmed at 5ūC per minute to 300ūC and then maintained at 300ūC for a further minute.
To follow the metabolic process, it was necessary to understand the basic nature of the fragmentation that was taking place in the mass spectrometer. In the electron ionization of the 17a-alkyl steroids the fragment pattern is dominated by the D-ring fragment ions. In the case of the fragmentation of 17a-methyltestosterone, the process involves the cleavage at the C13 to C17 bonds and at the C14 to C15 bonds.
This yields positively charged a, b-unsaturated ketone fragments at m/z 143. Consequently, observation of a pair of ions at m/z 143 and 146 in the EI mass spectra (the latter resulting from the labeled d3 steroids) is indicative that the D-ring is unaltered in the metabolic process.
Examples of chromatograms from the equine urine samples are shown in figure 59. The upper chromatogram represents the total ion current and the two, lower chromatograms were monitored using the specific ions having m/z values of 143 and 146 respectively.
Figure 59. Separation of the Metabolites of 17a-methyl-testosterone [after ref.8]