A stereochemical examination of the equine metabolism of 17alpha-methyltestosterone.

An investigation was conducted into the stereochemistry of the equine urinary metabolites of 17alpha-methyltestosterone observed after oral administration. Standards of the complete range of C3/C5/C16 stereoisomeric 17alpha-methylandrostane-3,17beta-diols, 17alpha-methylandrostane-3,16,17beta-triols and 17alpha-hydroxymethylandrostane-3,17beta-diols were purchased or synthesised, and were used to unequivocally identify the absolute structures of the metabolites. Phase I metabolism was found to involve combinations of Delta(4)-3-ketone reduction with both 5alpha,3beta- and 5beta,3alpha-stereochemistry, hydroxylation at C16 with both 16alpha- and 16beta-stereochemistry and hydroxylation of the 17alpha-methyl substituent. Phase II metabolism involved mainly sulfation with a lesser degree of beta-glucuronidation.

Selective manipulation of steroid hydroxyl groups with boronate esters: Efficient access to antigenic C-3 linked steroid-protein conjugates and steroid sulfate standards for drug detection.

The temporary protection of 17alpha-alkyl-5alpha-androstane-3beta,16beta,17beta triols as boronate esters is an efficient method for their regioselective functionalisation. This has been applied to the synthesis of protein-steroid conjugates 7-10 suitable for the development of immunoassays targeting classes of steroids banned from competition in Australian horse racing and other sports. The synthesis of steroids sulfate conjugates 42 and 44 for use as reference standards is also reported.

Detection of 17alpha-hydroxyprogesterone caproate in equine plasma by gas chromatography/tandem mass spectrometry.

A method was developed for the analysis of the synthetic progestin 17alpha-hydroxyprogesterone caproate in equine plasma following its administration by intramuscular injection. The method employed a reversed-phase solid-phase extraction followed by enol-trimethylsilylation and analysis by gas chromatography/tandem mass spectrometry. The intact ester was detectable in the plasma for up to 2 weeks after a single therapeutic dose, and was found to be stable in equine whole blood for at least 2 months.

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.

The detection of modafinil and its major metabolite in equine urine by liquid chromatography/mass spectrometry.

A method has been developed for the detection of modafinil and its major metabolite, modafinil acid, in equine urine by solid-phase extraction and positive ion electrospray ionisation liquid chromatography/mass spectrometry. The method has been applied to the analysis of equine urine samples obtained after the oral administration of modafinil. Modafinil acid was the major component in the urine, and was detected up to 4 days post-administration. Unchanged modafinil was present at substantially lower concentrations, and was detected for only 24 hours.

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry.

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.

Detection of stanozolol and its metabolites in equine urine by liquid chromatography-electrospray ionization ion trap mass spectrometry.

The equine phase I and phase II metabolism of the synthetic anabolic steroid stanozolol was investigated following its administration by intramuscular injection to a thoroughbred gelding. The major phase I biotransformations were hydroxylation at C16 and one other site, while phase II metabolism in the form of sulfate and beta-glucuronide conjugation was extensive. An analytical procedure was developed for the detection of stanozolol and its metabolites in equine urine using solid phase extraction, acid solvolysis of phase II conjugates and analysis by positive ion electrospray ionization ion trap LC-MS.

The detection of piroxicam, tenoxicam and their metabolites in equine urine by electrospray ionisation ion trap mass spectrometry.

An investigation has been conducted into the metabolism and urinary excretion of orally administered piroxicam and tenoxicam in the horse. The major component detected in urine after the administration of piroxicam was 5'-hydroxypiroxicam, which was detectable up to 24 h post-administration. Unchanged piroxicam was present only as a minor component. In contrast, unchanged tenoxicam was the major component observed after the administration of tenoxicam, being detectable for 72 h post-administration, while 5'-hydroxytenoxicam was a minor component. Phase II beta-glucuronide conjugation in each case was found to be negligible. The ion trap mass spectral characteristics of piroxicam, tenoxicam, 5'-hydroxypiroxicam and 5'-hydroxytenoxicam under electrospray ionisation conditions were examined in some detail.