亚胺连接的多孔共价有机骨架材料结合固相萃取-液相色谱-串联质谱检测蜂蜜中雌激素

以亚胺连接的多孔共价有机骨架材料(IL-COF-1)作为固相萃取的吸附剂,建立了液相色谱-串联质谱快速检测蜂蜜样品中痕量雌激素的方法。该研究选择雌二醇、己烯雌酚、雌三醇、β-雌二醇和炔雌醇5种雌激素作为目标分析物。在蜂蜜样品中添加雌激素,采用单因素优化法对影响萃取效果的重要因素进行优化,获得最佳条件:IL-COF-1用量为30 mg,样品流速为3 mL/min,样品溶液pH值为7,以5 mL的1%(v/v)氨水-甲醇溶液进行洗脱,流速为0.4 mL/min,萃取过程中不添加NaCl。采用高效液相色谱-三重四极杆质谱联用技术对提取物中的雌激素进行定量分析。以乙腈和5 mmol/L的乙酸铵溶液作为流动相进行梯度洗脱,经C18色谱柱分离,采用电喷雾离子源、质谱多反应监测和负离子扫描模式,实现了蜂蜜样品中5种雌激素的快速定性定量分析。在最佳条件下,方法验证结果中雌三醇、β-雌二醇和炔雌醇的线性范围为1~500 ng/g,雌二醇和己烯雌酚的线性范围为0.1~100 ng/g,相关系数(r)为0.9934~0.9972。检出限(S/N=3)为0.01~0.30 ng/g,定量限(S/N=10)为0.05~0.95 ng/g。添加50 ng/g 5种雌激素进行重复性实验,日内精密度相对标准偏差(RSD)为3.2%~6.6%,日间精密度RSD为4.2%~7.9%。基于IL-COF-1的固相萃取-液相色谱-串联质谱法具有快速准确、灵敏度高等特点,适用于蜂蜜中雌激素的分析和检测。将该方法应用于4个实际蜂蜜样品中雌激素的检测,均未检出目标物;在低中高3个水平下,5种雌激素的加标回收率为80.1%~115.2%,结果令人满意。

Imine-linked porous covalent organic framework used for the solid-phase extraction of estrogens from honey prior to liquid chromatography-tandem mass spectrometry

This study aimed to establish a method for the rapid determination of trace estrogens in honey samples by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using imine-linked porous covalent organic framework material (IL-COF-1) as the adsorbent for solid-phase extraction (SPE). Estradiol (E₁), diethylstilbestrol (DES), estriol (E₃), β-estradiol (E₂), and ethinylestradiol (EE₂) were used as the target analytes. A single factor optimization method was performed to optimize the extraction effect by adding estrogens to honey samples. The optimal conditions were as follows. A total of 30 mg IL-COF-1 was filled in the SPE column. The sample pH was adjusted to 7. The sample was loaded at a flow rate of 3 mL/min and eluted with 5 mL of a 1% (v/v) NH₃·H₂O-methanol solution. The flow rate of the eluent was 0.4 mL/min. NaCl was not added in the extraction process. HPLC coupled to electrospray ionization and triple quadrupole mass spectrometry was introduced to quantify the estrogens in the extracts. The estrogens were separated on a Thermo Fisher Scientific C18 analytical column (100 mm×2.1 mm, 5 μm). Acetonitrile and 5 mmol/L ammonium acetate solution were used as the mobile phases for gradient elution. The column temperature was set at 40 ℃, and the autosampler temperature was maintained at 10 ℃. The rapid qualitative and quantitative analysis of the five estrogens in the honey samples was operated under multiple reaction monitoring mode in a negative electrospray ion source mode. IL-COF-1 prepared in six batches was used as a filler for the SPE column. The relative standard deviations (RSDs) of the recoveries of the estrogens among different batches were 5.2%-9.1%. The reusability of IL-COF-1 material was assessed. After six SPE cycles on the same solid-phase extraction column, the RSDs of the estrogen recoveries were 2.5%-6.1%, indicating that IL-COF-1 has good reusability. The recoveries of estrogens obtained on an IL-COF-1 solid-phase extraction column within 6 days (tested once a day) were 95.1%-107.4%, and the RSDs were 6.2%-8.9%. These results confirmed that the SPE filler had good stability. The method validation results showed that the linear detection ranges were 1-500 ng/g for E₃, E₂, and EE₂, and 0.1-100 ng/g for E₁ and DES withe the correlation coefficients of 0.9934-0.9972. The limits of detection (LODs, S/N=3) were 0.01-0.30 ng/g, and the limits of quantification (LOQs, S/N=10) were 0.05-0.95 ng/g. Five estrogens were added (50 ng/g) for the repeated experiments. The RSDs of the intra-day precision were 3.2%-6.6%. The RSDs of the inter-day precision were 4.2%-7.9%. This method was applied to determine the estrogen levels in four honey samples, and no estrogen was found. The recoveries of the five estrogens in sample spiked at three levels including low, middle, and high levels were investigated, and satisfactory recoveries (80.1%-115.2%) were obtained. The SPE-HPLC-MS/MS method based on IL-COF-1 is rapid, accurate, and sensitive, making it suitable for analyzing and detecting estrogen in honey. Further exploration of the use of IL-COF-1 for the extraction processes is in progress.