10种麻痹性贝类毒素的固相萃取及液相色谱-串联质谱测定法

Detemination of 10 paralytic shellfish poisoning toxins by SPE and liquid chromatography-tandem mass spectrometry

建立了10种麻痹性贝类毒素（PSP）的高效液相色谱-串联质谱检测方法。样品用0.5%甲酸溶液加热提取,提取液经乙酸乙酯、三氯甲烷和HLB固相萃取柱净化,乙腈去蛋白后,在正离子模式下以电喷雾串联质谱仪进行测定,TSK改性凝胶柱为色谱分离柱,5 mmol·L-1甲酸铵（含0.5%甲酸）-乙腈（含0.5%甲酸）为流动相,采用基质匹配的标准曲线进行定量,并以虾夷扇贝（Patinopecten yessoensis）和菲律宾蛤仔（Ruditapes philippinarum）为测试对象,对方法的有效性进行了评价。评价结果为：在50～600μg·kg-1添加水平下,10种麻痹性贝类毒素平均回收率为72.3%～91.1%（批内）,相对标准偏差为3.9%～9.5%（批内）。STX、dc STX和neo STX定量限（S/N〉10）为100μg·kg-1,GTX1～GTX5、dc GTX2和dc GTX3定量限为50.0μg·kg-1。结果表明,该方法快速、准确,适合于贝类产品中麻痹性贝类毒素的测定。

Paralytic shellfish poisoning （PSP） toxin is a neurotoxin that can cause prpalytic poisoning when people eat shellfish containing such toxins. PSP is the most harmful marine biotoxin with the highest accident frequency, meanwhile it is the most widely distributed biotoxin globally. At present, three methods are mainly used for the determination of PSP, including enzyme-linked immune method, biological method, liquid chromatography with fluorescence method and liquid chromatography-tandem mass spectrometry （LC-MS/ MS）. LC- MS/MS is a newly developed technology for detecting PSP. Sample pretreatment and chromatographic separation are the crucial procedures with certain difficulty in the determination of PSP for its special chemical properties and the presence of isomers. Although there are some research results reported both at home and abroad, the application is limited for the low extraction efficiency, poor purification effect and the lack of detection of Saxitoxin with high toxicity. In the present study, 10 kinds of common PSP （ GTX1, GTX2, GTX3, GTX4, GTX5, dcGTX2, dcGTX3, dcSTX, neoSTX and STX） with high toxicity were chosen as target eompouds. By studying sample pretrcatment method, screening of chromatographic column, optimizing of mobile phase gradient and acidity, we successfully solved the effect of water-soluble protein on the influence of liquid chromatography -mass spectrometry system and the influence of acidity on the retention time, and improved the sensitivity of the method. The sample was extracted with 0.5% formic acid in 100 ℃ water. The extract was cleaned up by ethyl acetate, chloroform and HLB column, and analyzed by HPLC-MS/MS in ESI ＋ after elimination of protein by acetonitrile, and the TSK-Gel Amide-80 column was used for seperation, and the mobile phase was 5 mmol ·L-1 ammonium formate with 0.5 % formic acid-acetonitrile with 0.5% formic acid. The concentration of target compounds was quantified by matrix-matched calibration curve. In the experiment, we took Ruditapes philippinarum and Crassostrea gigas as matrixes to evaluate the validity of the method, and results showed that the average recoveries intra-assay ranged from 72.3% to 91.1% in the spiked range of 50 -600μg · kg-l. The relative standard deviations intra-assay ranged from 3.9% to 9.5%. The limits of quantification （ S/N 〉 10） were 100μg · kg-1 for STX,dcSTX and neoSTX, 50μg· kg-i for GTX1-GTX5, dcGTX2 and dcGTX3 respectively. The results indicate that the method developed is faster, more accurate, and is suitable for PSP detection in shellfish.