Determination of two pancreatic stimulants in equine urine using ionic liquid-based deep eutectic solvent-assisted liquid-liquid microextraction and ultra-high performance liquid chromatography-tandem mass spectrometry

Acetohexamide （AH） and chlorpropamide （CP） can stimulate pancreatic secretion of insulin and regulate water-salt balance， which may indirectly lead to an increase in adrenaline and potentially enhance the athletic performance of horses. For the protection of horse welfare and the consideration of fair competition， AH and CP were listed as pancreatic stimulants by the Federation Equestre Internationale （FEI）. A method for the detection of pancreatic stimulants in equestrian sports needs to be established. Before analyzing these drugs from biological samples such as urine， it is necessary to perform pre-treatment to separate and concentrate the target analytes. However， there are several disadvantages to using traditional pre-treatment methods， including toxicity and inefficiency. A rapid and environmentally friendly pre-treatment method utilizing ionic liquid-based deep eutectic solvent vortex-assisted liquid-liquid microextraction （IL-DES-VALLME） was developed. Coupled with ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）， the approach enabled efficient extraction and sensitive quantification of AH and CP from equine urine. <italic>N-</italic>Butyl-<italic>N-</italic>methylmorpholinium bis（（trifluoromethyl）sulfonyl）imide （EMMPNTF₂） was chosen as the hydrogen bond acceptor （HBA）， combined with pentafluorophenol （PFP） acting as the hydrogen bond donor （HBD）. A molar ratio of 1∶4 （HBA/HBD） was identified as the optimum composition of IL-DES. Differential scanning calorimetry （DSC） was utilized to measure the melting point of the synthesized IL-DES. Fourier-transform infrared （FT-IR） spectra and proton nuclear magnetic resonance （¹H NMR） spectra were employed to characterize the structure of the IL-DES and investigate the hydrogen bonding interactions between its components. Under the optimum conditions， only 100 μL of IL-DES， 1 mL of equine urine， and 6 min were required. An external standard calibration method was utilized for quantification， and a linear relationship was achieved with a mass concentration range of 0.2-20 000 ng/mL for AH and 0.5-20 000 ng/mL for CP. The coefficient of determination （<italic>r</italic>²） were all greater than 0.999. The limits of detection （LOD） of the method based on a signal-to-noise ratio of 3 were 0.1 ng/mL for AH and 0.2 ng/mL for CP. The limits of quantification （LOQ） of the method based on a signal-to-noise ratio of 10 were 0.2 ng/mL for AH and 0.5 ng/mL for CP. The absolute values of matrix effects were all less than 20% （-8.9% for AH， -11.2% for CP）. Method accuracy， assessed through recovery studies at multiple concentrations across the linear range， yielded values between 90.2% and 110.4%. Precision， expressed as relative standard deviation （RSD）， was excellent， with intra-day RSDs ≤ 6.6% and inter-day RSDs ≤ 5.8%. The RSDs for robustness were less than 5.7%， and the average carry-over ratios were in the range of 11.4%-17.3%， indicating good method robustness and acceptable carry-over levels for the quantitation of AH and CP in equine urine. The greenness was assessed by the AGREEprep metric （score： 0.63）. Compared with traditional extraction methods， the IL-DES-VALLME method has the advantages of rapidity， simplicity， efficiency， low toxicity， and low cost. Additionally， the application of the proposed approach to real equine urine samples proved its practicability. These advantages make the method very appealing for doping control in equestrian sports.