Anabolic-androgenic steroids (AASs) are banned in equine sports owing to their performance-enhancing properties. Conventional gas chromatography-based analytical techniques require extensive sample preparation. This study synthesised commercially unavailable AAS sulfate metabolites and developed an UHPLC-QTOF-MS method for direct qualitative screening of intact sulfo-conjugated AASs (sulfo-AASs) in equine urine.
Eighteen sulfo-AASs were synthesised and analysed by UHPLC-QTOF-MS using a C18 column with elution gradient: 0-0.50 min (10%B), 0.51-20.50 min (10-100%B), 20.51-23.00 min (100%B), 23.01-27.50 min (10%B). Mobile phases: 5 mM ammonium formate in water (A) and in methanol (B). Untargeted data-dependent acquisition (DDA) method in negative electrospray ionisation mode confirmed and characterised the synthesised sulfates. Targeted screening method development involved spiking blank gelding urine, solid-phase extraction on weak-cation exchange cartridges, and product-ion scan mode under the same LC-MS conditions.
Most sulfo-AAS compounds were detected as mono-sulfate conjugates with distinct mono-chromatographic peaks and retention times. Endogenous steroids with diol functional groups produced additional peaks, indicating positional isomers from multiple sulfation reactions. Matrix effect, recovery, and process efficiency (PE) assessments indicated widespread ion suppression (<100%) and variable recoveries (20% to 90%), with endogenous diol sulfates showing the lowest recovery (18% - 31%). PE was consistently lower for the endogenous compared to exogenous sulfo-AASs, particularly with the estrane class, including exogenous trenbolone sulfate. Reduced chromatographic resolution was observed only for DHEA sulfate and 1-testosterone sulfate.
DDA-derived retention time, exact mass, and fragmentation data supported the development of a targeted UHPLC-QTOF-MS method. With SPE, most sulfo-AASs were detectable at 10 ng/mL. This demonstrates the feasibility of direct detection of intact phase II metabolites and highlights the potential that the same approach can be employed to detect structurally related compounds.
In conclusion, this method streamlines sample preparation and demonstrates potential for routine implementation in anti-doping laboratories to achieve faster, more cost-effective, and comprehensive testing.