Simultaneous Determination of 4 Eugenol Derivatives Anesthetics Residues in Aquatic Products by UPLC-MS/MS

The method for simultaneous determination of 4 eugenol derivatives anesthetics residues in aquatic products was developed by solid phase extraction coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The influences of extraction procedures, chromatographic conditions and mass spectrometry conditions were investigated. The optimal solid phase extraction experiment conditions were as follows: the aquatic samples were extracted with 5 mL hexane twice, supernatant was combined and centrifuged at 6 000 r/min, enriched by nitrogen blowing at 35 ℃, mixed with 3 mL 40% methanol, cleaned up by solid phase column of hydrophilic-lipophilic balance (HLB) cartridge, then rinsed by 3 mL 40% methanol, eluted by 2 mL methanol, dried with nitrogen gas at 35 ℃. The chromatographic separation was performed within 13.0 min on an Agilent ZORBAX Eclipse Plus C18 column （50 mm×2.1 mm×1.8 μm）. Formic acid played a key role in mobile phase as modifier to improve the ionization efficiency of methyleugenol and methylisoeugenol. The flow rate was 0.30 mL/min. An Agilent 6470 Triple Quadrupole mass spectrometer (QQQ-MS) was operated in the positive and negative ion modes at the same time using multiple reaction monitoring （MRM） for qualitative and quantitative analysis of 4 eugenol derivatives which were quantitated with an external standard method. The QQQ-MS conditions were optimized including ionization mode, precursor ion, product ion, fragmentor voltage, and collision energy. The limits of quantitation for eugenol and isoeugenol are 2.5 μg/kg while the limits of quantitation for methyleugenol and methylisoeugenol are 1.0 μg/kg. The linear correlation coefficient (R²) is over 0.996 in the range of 1.200 μg/L for eugenol derivatives’ concentrations. The average recoveries for eugenol derivatives are 77.6%-111.4% with RSDs of 2.7%-9.1%. This method is precise, selective and reliable, and has been successfully applied for detection and determination of eugenol, isoeugenol, methyleugenol and methylisoeugenol simultaneously in batch sample of aquatic products.