UHPLC MS/MS分析腹泻性贝毒在文蛤体内的积累与排出

采用超高压液相色谱-四极杆串联质谱法建立了腹泻性贝毒(diarrhetic shellfish poisoning,DSP)、大田软海绵酸(okadaicacid,OA)和鳍藻毒素1(dinophysistoxin-1,DTX1)的检测方法。样品经80%甲醇提取后,经正己烷脱脂和C18固相萃取柱净化,采用Hypersil GOLD C18色谱柱分离,以50%(体积分数)乙腈水(0.2%甲酸)溶液为流动相等度洗脱,电喷雾电离,在选择反应监测(SRM)模式下进行定性与定量分析。OA和DTX1在25～400μg/kg范围内线性关系良好,OA和DTX1最低定量限(LOQ,S/N>10)分别为5μg/kg和1μg/kg。样品平均回收率大于80%,相对标准偏差小于10%(n=6)。应用该检测方法研究了利玛原甲藻(Pivrocentrurn lima)产生的DSP毒素在文蛤(Meretrixmeretrix)体内累积与排出规律。结果表明,DSP在文蛤体内积累迅速,摄食48 h,贝类肌肉中和消化腺中的DTX1含量即超过了食用安全限量。文蛤消化腺中的毒素含量大约是肌肉中的10倍。经96 h排出实验,贝体内的DSP毒素排除量大于80%。

UHPLC MS/MS analysis of the accumulation and elimination of DSP in Meretrix meretrix

Okadaic acid(OA) and dinophysistoxins-1(DTX1) of diarrhetic shellfish poisoning(DSP) toxins were detected by ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry(UHPLC-MS/MS).After the toxins being extracted using methanol and water(80∶ 20,V/V),the solution was further extracted with n-hexane to remove lipid components and then cleaned up by solid phase extraction(SPE) on an C18 cartridge.The analytes were eluted with 50%(V/V)acetonitral water containing 0.2% formic acid on a Hypersil GOLD C18 column.The qualitative and quantitative determination of the toxins was performed by selective reaction monitoring(SRM) mode.OA and DTX1 were determined in the negative ion mode.The calibration curve was linear in the ranges of 25-400 μg/kg.The quantification limits of OA and DTX1were 5 μg/kg and 1 μg/kg,respectively.The mean recoveries at spiked concentrations of 5-100 μg/kg were more than 80% and the relative standard deviations were less than 10%(n = 6).We studied toxin accumulation,transformation and elimination of DSP in Meretrix meretrix.The results showed that the increase in DSP content with the increase in time both in digestive gland and muscle during an 48-hour accumulation period when Meretrix meretrixs were fed with P.lima.At the end of the accumulation period,the toxin amount of DTX1 in Meretrix meretrix reached the regulatory limit in shellfish for OA group toxins.After the 96-hour elimination period,the amount of DSP decreased at least by 80%.