在1.0 g充分匀质的样品中加入80%(体积分数,下同)甲醇溶液4 mL,涡旋2 min,超声10 min,冷冻(4℃)离心5 min,重复提取一次。合并上清液,用80%甲醇溶液稀释至10 mL。分取5 mL,加入20 mL磷酸盐缓冲液(pH 7.3),涡旋混匀。上述溶液过活化好的免疫亲和柱,用20%(体积分数)甲醇溶液3 mL淋洗柱子,加压抽干柱子后再加入甲醇3 mL洗脱,净化过程中控制温度为室温,过柱速率为2~3 mL·min^-1。收集洗脱液,分取1.0~1.5 mL,离心10 min,上清液用超高效液相色谱-三重四极杆复合线性离子阱质谱法测定。以Poroshell 120 EC-C₁₈色谱柱为固定相,以不同体积比的10 mmol·L^-1甲酸铵-0.1%(体积分数)甲酸溶液和甲醇的混合溶液为流动相进行梯度洗脱,用多反应监测-信息依赖性分析-增强子离子(MRM-IDA-EPI)扫描模式获得定量结果,并同时完成目标物二级质谱图的匹配。结果显示:短裸甲藻毒素的质量浓度在5~500 μg·L^-1内与其对应的峰面积呈线性关系,检出限(3S/N)为30 μg·kg^-1;对阴性样品进行加标回收试验,回收率为80.5%~108%,测定值的相对标准偏差(n=6)为3.1%~6.0%。方法用于双壳贝类样品的分析,未检出短裸甲藻毒素,而加标样品中目标物的EPI二级质谱图与标准二级质谱图的匹配度值、反匹配度值和纯度值均达到60%以上,说明目标物和短裸甲藻毒素相似度较高。

Fully homogenized sample (1.0 g) was extracted with 4 mL of 80% (volume fraction, the same below) methanol solution, whirled for 2 min, sonicated for 10 min, and centrifuged freezingly for 5 min at 4 ℃, and the extraction was made once again. The supernatant was combined, and diluted to 10 mL with 80% methanol solution. An aliquot (5 mL) was taken and mixed with 20 mL of phosphate buffer solution (pH 7.3). After vortexing, the above solution was passed through the immunoaffinity column activated beforehand, and the column was rinsed with 3 mL of 20% (volume fraction) methanol solution, drained under pressure, and eluted with 3 mL of methanol. During the purification process, the temperature was controlled at room temperature, and the column passing rate was set to 2-3 mL·min^-1. The eluate was collected, and an aliquot (1.0-1.5 mL) was centrifuged for 10 min. The supernatant was determined by ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry. Poroshell 120 EC-C₁₈ column was used as the stationary phase, and gradient elution was performed with the mixture of 10 mmol·L^-1 ammonium formate-0.1% (volume fraction) formic acid solution and methanol at various volume ratios as mobile phase. Multiple reaction monitoring-information dependent acquisition-enhanced production ion (MRM-IDA-EPI) scanning mode was used for obtaining the quantitative results and completing the matching of secondary mass spectrum. It was shown that the linear relationship between values of the peak area and the mass concentration of brevetoxin was kept in the range of 5-500 μg·L^-1, with detection limit (3S/N) of 30 μg·kg^-1. Test for recovery was made on the negative sample by standard addition method, giving recoveries in the range of 80.5%-108%, and RSDs (n=6) of the determined values ranged from 3.1% to 6.0%. The proposed method was applied to the analysis of bivalves, and brevetoxin was not detected. Values of the matching degree, reverse matching degree and purity between EPI secondary mass spectrum of the target in the spiked sample and standard secondary mass spectrum were above 60%, and it was turned out that a high similarity was obtained between the target and brevetoxin.