Investigation of the metabolism of anabolic steroids in racing greyhounds using liquid chromatography/electrospray mass spectrometry

An in vitro canine liver microsomal assay was developed for the purpose of generating metabolites from the anabolic steroids testosterone, methyltestosterone, mibolerone, and boldenone. Identification of their in vitro biotransformation products was used to predict possible in vivo metabolites typically extracted from urine of racing greyhounds. The microsomal assay was optimized for incubation time and volume as well as substrate and protein concentration. Metabolite identifications were based on interpretation of chromatographic retention, ultraviolet spectral, mass spectral M+1 ions, and source induced dissociation mass spectral fragmentation data. Analysis of deuterium labeled, $\sp{13}$C labeled, and structurally related steroids allowed for the identity and possible fragmentation mechanisms of the primary source induced fragment ions (m/z 97, m/z 109, and m/z 123) of testosterone. Metabolites were identified in beagle and greyhound microsomes to assess breed differences in steroid biotransformation. Primary beagle microsomal metabolites were identified as androstenedione and 6$\beta$-hydroxytestosterone for testosterone, 6$\beta$-hydroxymethyl-testosterone and a monohydroxymethyltestosterone for methyltestosterone, two monohydroxymibolerones for mibolerone, and a monohydroxyboldenone, an oxidized boldenone, and testosterone for boldenone. Primary greyhound microsomal metabolites were identified as androstenedione and 16$\alpha$-hydroxytestosterone for testosterone, 6$\beta$-hydroxymethyltestosterone and two monohydroxymethyltestosterones for methyltestosterone, two monohydroxymibolerones for mibolerone, and three monohydroxyboldenones, an oxidized boldenone, testosterone, and a boldenone isomer for boldenone. Beagle microsomes appear to be a good model to predict in vitro greyhound steroid biotransformation products. Greyhound administration studies were performed to determine the predictability of the in vitro microsomal assay for urinary metabolites and to assess temporal excretion profiles of these investigated steroids. Primary urinary metabolites for testosterone were identified as a ketotestosterone, a testosterone isomer, and a reduced monohydroxytestosterone. Primary urinary metabolites for methyltestosterone were identified as a reduced methyltestosterone, an oxidized monohydroxymethyltestosterone, and a reduced monohydroxymethyltestosterone. Four monohydroxymibolerones were the primary urinary metabolites for mibolerone. One of the monohydroxymibolerones was consistent with a metabolite identified in both the beagle and greyhound microsomal assays. Primary urinary metabolites for boldenone were identified as an oxidized monohydroxyboldenone, a monohydroxyboldenone, and two reduced monohydroxyboldenones. Minimal correlates existed between the metabolites generated in vitro and in vivo. The discrepancies are likely due to the limitations of the microsomal system.