Simultaneous Determination of Eugenol and Its Five Derivatives in Aquatic Products by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry Combined with Quick, Easy, Cheap, Effective, Rugged and Safe Extraction

An ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for the simultaneous determination of eugenol and its five derivatives including isoeugenol, methyl eugenol, methyl isoeugenol, acetyl eugenol and acetylisoeugenol in aquatic products. The samples were extracted with acetonitrile, and then the extracted supernatant was cleaned up with 50 mg of primary secondary amine (PSA) and 100 mg of C₁₈ and 150 mg of MgSO₄. Separation of the targets was performed on a Kinetex C₁₈ (2.1 mm × 100 mm, 2.6μm) chromatographic column by gradient elution using a mobile phase of water and methanol. The six eugenol derivatives were detected using an electrospray ionization (ESI) source with fast switching between the positive and negative ionization modes in the multiple reaction monitoring (MRM) mode, and quantified by the internal standard method using eugenol-D₃ as the internal standard (IS). The calibration curves showed a good linearity in the range of 2–100 ng/mL for eugenol and isoeugenol, and 1–100 ng/mL for four other eugenol derivatives with correlation coefficient (r) ≥ 0.999. The average recoveries of spiked samples were between 67.0% and 106.4% with relative standard deviations (RSDs) of 1.2%–15.7%. The limits of detection (LOD, R_SN = 3) and quantitation (LOQ) were 0.5–1.5 and 2.0–5.0 μg/kg, respectively. The method is an accurate, rapid and reliable assay for the detection of eugenol and its derivatives in aquatic products.