Abstract:

Due to the harmful effects of estrogens and their prevalence in animal foods, accurate analysis of estrogen levels in animal foods is imperative in order to effectively assess food safety risks and ensure consumer safety. Therefore, a rapid and accurate method based on PRiME HLB solid phase extraction (SPE) cartridge purification and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine nine estrogen residues in bullfrogs. The nine estrogens included estriol (E3), 17β-estradiol (β-E), 17α-estradiol (α-E), 17α-ethinylestradiol (EE2), estrone (EI), diethylstilbestrol (DES), dienestrol (DE), hexestrol (HEX), and dienestrol diacetate (DD). This study optimized the mobile phase system, extraction solvent, and SPE cartridges. Because estrogens present weak alkalinity, adding a small amount of alkaline substance to the mobile phase benefits estrogen ionization into the ionic state, eliminates the peak trailing phenomenon, and enhances the signal response of estrogens to improve sensitivity. Estrogens have one or more hydroxyl groups in their chemical structures. According to the principle of similar solubility, polar solvents are chosen as extraction solvents. Based on the complex matrix composition of meat samples, SPE is required for purification to reduce matrix effects. The liquid chromatographic conditions were optimized, and the 0.5 mmol/L ammonium fluoride aqueous solution-acetonitrile system as mobile phases showed better sensitivity than the ammonium acetate aqueous solution-acetonitrile system and the ammonia-acetonitrile system for the nine estrogens. When acetonitrile was used as the extraction solvent, the extraction rates of all nine estrogens exceeded those of methanol and ethyl acetate and increased by 15%-40%. By focusing on the matrix purification effect of four different SPE cartridges, the results showed that the matrix purification ability of the PRiME HLB cartridge outperformed that of the HLB, C₁₈, and Silica SPE cartridges. After purification by the PRiME HLB cartridge, the recoveries of all compounds were in the range of 70%-125%, and the DD recovery was increased from 47% to 74%, whereas the HEX recovery was reduced from 180% to 123%. Therefore, the PRiME HLB SPE cartridge was selected as the cleanup material for this experiment. Finally, the sample was extracted using acetonitrile, purified by PRiME HLB SPE cartridge, and separated on a Waters Acquity UPLC BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm) with a mobile phase of 0.5 mmol/L ammonium fluoride aqueous acetonitrile solution at a flow rate of 0.3 mL/min. The detection was conducted in positive and negative ion switching mode (ESI⁺/ESI^-) and multiple reaction monitoring (MRM) scanning, and it was quantified using a matrix-matched external standard method. Under the optimal experimental conditions, the linear ranges were 0.5-100.0 μg/L for E3, β-E, α-E, EI, DE, HEX, and DD, and 1.0-100.0 μg/L for EE2 and DES. The nine estrogens showed good linearity in all linear ranges, with correlation coefficients of 0.9953-0.9994. The limits of detection were 0.17-0.33 μg/kg, and the limits of quantification were 0.5-1.0 μg/kg. The recoveries of the nine estrogens spiked at the three spiked levels of low (2.0 μg/kg), medium (10.0 μg/kg), and high (80.0 μg/kg) were 107.4%-125.3%, 67.0%-123.3%, and 65.1%-128.2%, respectively. The relative standard deviations were 1.9%-17.6%. The method established in this study was applied to detect nine estrogen residues in 50 commercially available bullfrog samples, and the results showed that HEX, EI, and DES were detected in few samples. The method is simple, rapid, sensitive, and reproducible, and can be used for the simultaneous, rapid and accurate determination of large quantities of samples.

Key words: solid phase extraction (SPE), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), estrogen, bullfrog