Fig. 1. The common D-ring structure which arises from the metabolism of 17-alkyl anabolic steroids.

Fig. 2. Representative 17-alkyl steroids 1–4 and examples of their corresponding metabolites 5–8.

Fig. 3. Steroids conjugated to HSA for immunoassay development.

Fig. 4. Validation of the cross-reactivity comparisons: parallelism of various cross-reacting steroid ligands on the 16β-hydroxymestanolone plate. [Parent compound is 16β-hydroxymestanolone (9). 100% = 16β-hydroxystanozolol, 84% = 16β-hydroxymethandrenone, 40% = 16α-hydroxyfurazabol, 5.5% = 19-nor-5α,17β-pregnane-3β,16β,17β-triol and 0.963% = stanozolol].

Fig. 5. Validation of the cross-reactivity comparisons: parallelism of various cross-reacting steroid ligands on the mestanolone plate. [Parent compound is mestanolone (10). 100% = methandriol, 60.3% = oxymetholone, 34.4% = ethisterone, 16.5% = danazol and 0.631% = estrone].

Fig. 6. Excretion curves, determined using the 16β-hydroxymestanolone ELISA plate, detecting stanozolol metabolites in equine urine, as shown by the absorbance suppression relative to the negative control. Following the intramuscular injection of 250 mg of stanozolol, the analyses were performed on urine which was: (a) hydrolysed with β-glucuronidase (1/100 dilution), (b) subjected to reverse-phase SPE, methanolysed and quenched and (c) subjected to mixed-mode SPE to give a basic eluate.

Fig. 7. Excretion curves, determined using the mestanolone ELISA plate, detecting methandriol metabolites in equine urine, as shown by the absorbance suppression relative to the negative control. Following the intramuscular injection of 375 mg of methandriol dipropionate, the analyses were performed on urine which was (a) subjected to reverse-phase SPE, then methanolysed and quenched and (b) treated via mixed-mode SPE to give a neutral eluate.

Fig. 8. Excretion curves, determined using a 16β-hydroxymestanolone ELISA plate, detecting methandriol metabolites in equine urine, as shown by the absorbance suppression relative to the negative control. Following the intramuscular injection of 375 mg of methandriol dipropionate, the analyses were performed on urine which was (a) subjected to reverse-phase SPE, then methanolysed and quenched and (b) treated via mixed-mode SPE to give a neutral eluate.

Fig. 9. Excretion curve, determined by GCMS analysis, for methandriol in equine urine following the intramuscular injection of 375 mg of methandriol dipropionate. The analysis was performed on urine subjected to reverse-phase SPE, methanolysis and quenching, followed by derivatisation with pentafluoropropionic anhydride.

Fig. 10. Synthesis of 16β-hydroxymestanolone (9).

Fig. 11. Conjugation of steroids to HSA via two different peptide-coupling methods: direct carbodiimide activation or N-hydroxysuccinimide ester activation.

Fig. 12. Anabolic steroids with high cross-reactivities in the 16β-hydroxymestanolone assay.

Fig. 13. Anabolic steroids administered to horses for analysis of equine urine via the 16β-hydroxymestanolone and mestanolone assays.

Molar ratios (steroid/HSA) determined by UV analyses at 254 nm

Antibody titres determined for four HSA conjugates 87 days after primary vaccination

Percentage cross-reactivities for anabolic steroids standards: 16β-hydroxymestanolone plate with antibodies produced against human serum albumin-conjugate (24)

Percentage cross-reactivities for anabolic steroids standards: mestanolone plate with antibodies produced against human serum albumin-conjugate (25)