We present a case of a mixed martial art athlete found positive to methasterone metabolites, ligandrol and cardarine metabolites after an anti-doping testing. The athlete denied any drugs consumption but provided the list of 23 nutritional supplements under use during competition. The aim of the present study was to analyse the provided material to support evidence of unintentional doping. Cumulatively, products were commercialised either as pre-workout, intra-workout and post-workout adjuvants containing amino acids, creatine and proteins or as cofactors and vitamins to recover after training. Four products were taken to aid sleeping and one was a “testosterone boost”.

All samples were submitted to analysis by liquid-chromatography/high resolution mass spectrometry (LC-QTOF). One hundred milligrams of material (powders, pills, oily pills, capsules) were sampled and dissolved with 10mL of methanol, vortexed for 30 and sonicated for 30min. The extracts were centrifuged at 3000rpm for 5min at 25°C. The supernatants were appropriately diluted with deionised water then 5μL injected into the LC-MS. Separations were carried out in a Zorbax C18 (2.1×100, 1.8μm) column in 5mM ammonium formate (mobile phase A) and methanol (mobile phase B), both added of 0.1% formic acid, flow at 0.2mL/min. Acquisitions were carried out in positive polarity MS scan mode and MS/MS for fragment confirmation.

Sensitivity expressed as method detection limit (MDL), linearity, carry-over, dilution integrity, accuracy and precision were tested under the described conditions for cardarine, methasterone and ligandrol. The analysis of the samples showed 2 out the 23 nutritional products containing the illicit substances. Methasterone, cardarine and ligandrol were alongside present in one sample (S1) at the respective concentrations of 5μg/mg, 11.8μg/mg and 1.4μg/mg. Cardarine and ligandrol were confirmed in a second sample (S2) at the concentration of 0.6 and 0.2μg/mg, respectively.

Two products resulted contaminated with methasterone, cardarine and ligandrol. The calculated concentration for all substances>1μg/mg in S1 product may presume an intentional manipulation with pharmacologically active ingredients that better fitted the promise of enhanced recovery and improved endurance. In S2 ligandrol and cardarine amounts are compatible with a cross-contamination scenario.

Conclusion: methasterone, ligandrol and cardarine were complessively detected in 2 samples. Since no labelling declared the retrieved substances, this might have represented a case of unintentional doping.