A hydrophobic deep eutectic solvent-based vortex-assisted liquid-liquid microextraction applied for doping control of aromatase inhibitors from equine urine.

Aromatase inhibitors (AIs) can indirectly cause increased testosterone in animals, which leads to the improvement of the athletic ability of horses. For the protection of horses and the consideration of fair competition, AIs were listed as prohibited drugs by the Federation Equestre Internationale (FEI). There were several disadvantages using traditional pretreatment methods before analyzing these drugs from biological samples. A rapid and green pretreatment method has been developed by utilizing the hydrophobic deep eutectic solvent (DES)-based vortex-assisted liquid-liquid microextraction (DES-VALLME) followed by ultra-high performance liquid chromatography tandem triple-quadrupole mass spectrometry (UHPLC-MS/MS) technique for the efficient extraction and sensitive detection of AIs in equine urine samples. The combination of menthol and 4-fluorophenol in a molar ratio 1:4 was chosen as the optimum composition of DES for extracting AIs. Under the optimum conditions, only 80 μL of DES, 1 mL equine urine and 2 min were expended. An external standard calibration method was utilized for determination, and a linear relationship was achieved with a concentration range of 0.02-4000 ng mL (r ≥ 0.9983). The limits of detection of the method based on a signal-to-noise ratio of 3 were 0.01-4 ng mL. The accuracy recoveries ranged from 94.9% to 113.4% within the intra-day and inter-day relative standard deviations of less than 9.1%. Compared with traditional extraction methods, the DES-VALLME method had the advantages of rapidity, simplicity, efficiency, low toxicity, and low cost. This method has potential and possessed brilliant prospects for doping control.