基于通过型固相萃取-超高效液相色谱-串联质谱法同时测定牛蛙中9种雌激素北大核心CSCD

建立了基于通过型固相萃取小柱净化的超高效液相色谱-三重四极杆质谱联用(UPLC-MS/MS)同时快速准确测定牛蛙中9种雌激素(雌三醇(E3)、17β-雌二醇(β-E)、17α-雌二醇(α-E)、17α-炔二雌醇(EE2)、雌酮(EI)、己烯雌酚(DES)、己二烯雌酚(DE)、己烷雌酚(HEX)、醋酸双烯雌酚(DD))残留的检测方法。样品经乙腈提取,经PRiME HLB固相萃取柱净化,Waters Acquity UPLC BEH C_(18)柱(100 mm×2.1 mm,1.7μm)分离,以0.5 mmol/L氟化铵水溶液-乙腈体系为流动相梯度洗脱,流速为0.3 mL/min,采用电喷雾正负离子切换模式(ESI^(+)/ESI^(-))和多反应监测(MRM)扫描方式检测,基质匹配外标法定量分析。该研究优化了液相色谱条件,相比于乙酸铵水溶液-乙腈体系和氨水溶液-乙腈体系,0.5 mmol/L氟化铵水溶液-乙腈体系作为流动相时9种雌激素普遍具有更佳的灵敏度。相比于甲醇和乙酸乙酯,乙腈作为提取溶剂时9种雌激素的提取率提高15%~40%。考察了HLB、C_(18)、Silica、PRiME HLB共4种不同类型的固相萃取小柱的基质净化效应,结果表明,PRiME HLB柱具有更好的基质净化能力。经PRiME HLB净化后,所有化合物的回收率均在70%~125%之间。DD的回收率从47%提高到74%,DES的回收率从180%降低到123%,有效减弱了基质效应。在最佳的实验条件下,E3、β-E、α-E、EI、DE、HEX、DD的线性范围为0.5~100.0μg/L,EE2和DES的线性范围为1.0~100.0μg/L,9种雌激素在各自的线性范围内均有良好的线性关系,相关系数为0.9953~0.9994,方法检出限为0.17~0.33μg/kg,方法定量限为0.5~1.0μg/kg,在2.0、10.0、80.0μg/kg 3个加标水平下,9种雌激素的加标回收率为65.1%~128.2%,相对标准偏差为1.9%~17.6%。该方法操作简便、快速、灵敏,重复性好,可用于大批量样品的同时快速准确检测。

Simultaneous determination of nine estrogens in bullfrogs using filtered solid phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry

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.