Page 197 - Tandem Techniques

Page 180

The mass spectrometer employed was the Finnigan MATTSQ 70 and the separation was carried out on
a BPX5 column 25 m long, 250 µm I.D. The column was operated isothermally at 150°C for one
minute, programmed at 5 °C per minute to 300°C and then maintained at 300°C for a further minute.

To follow the metabolic process, some knowledge of the basic nature of the fragmentation process that
takes place in the mass spectrometer is helpful. In the electron ionization of the 17a-alkyl steroids the
fragment pattern is dominated by the D-ring fragment ions. For the fragmentation of 17a-
methyltestosterone, the process involves the cleavage at the C to C17 bonds and at the C to C
15
14
13
bonds. This yields positively charged a, ß-unsaturated ketone fragments at m/z 143. Consequently,
observation of a pair of ions at m/z 143 and 146 in the El mass spectra (the latter resulting from the
labeled d steroids) is indicative that the D-ring is unaltered in the metabolic process.
3

Figure 5.11
Separation of the Metabolites
of 17a-methyltestosterone [ref.8]

Examples of chromatograms from the equine urine samples are shown in figure 5.11. The upper
chromatogram represents the total ion current and