石墨化炭黑固相萃取-亲水液相色谱-三重四极杆质谱法测定海产品中河豚毒素

Determination of tetrodotoxin in seafood using graphitized carbon black clean-up with hilic ultra performance liquid chromatography-triple quadrupole mass spectrometry

目的建立石墨化炭黑固相萃取-亲水液相色谱-三重四极杆质谱法测定海产品中河豚毒素的检测方法。方法将河豚鱼肉、肝和织纹螺肉样品经0．2％乙酸水溶液提取后（肝脏提取液需经HLB固相萃取柱脱脂），再经石墨化炭黑固相萃取柱净化，以含有2．0mmol／L甲酸铵-0．1％（体积分数）甲酸的95％（体积分数）乙腈水液及含2．0mmol／L甲酸铵的0．1％甲酸水溶液作为流动相进行梯度洗脱，在色谱柱上（AcquityUPLCBEHAmide。100mm×2．1mm×1．7μm）实现分离，正离子电喷雾多反应监测方式检测，基质标准外标法定量。结果河豚鱼肉、肝和织纹螺肉中TTx的线性范围分别为30～10000、50—10000和30～10000ng／kg，相关系数为0．9963～0．9990；检出限分别为10、20和10μg／b，定量限分别为30、50和30μg/kg；平均加标回收率分别为81．5％～93．1％、82．3％～106．0％和83．5％～95．2％，RSD分别为2．3％～11．0％、4．3％-14．0％和3．5％～13．0％（n=6）。结论本法简单、准确、灵敏，可应用于河豚鱼和织纹螺中TTX的测定。

Objective To develop a rapid hilic ultra performance liquid chromatography （UPLC）- mass spectrum（ MS）/MS method for determination of tetrodotoxin in seafood. Methods The sample of muscle and liver of puffer fish and nassarius were extracted with aqueous solution containing 0. 2% （V/V） acetic acid （ the extract of liver must be purified through HLB cartridge）, and then cleanup of extract was accomplished by solid-phase extraction with a graphitized carbon black cartridge. The analysis of tetrodotoxin was carried out on a chromatographic column （Acquity UPLC BEH Amide, 100 mm × 2. 1 mm × 1.7 μm） with gradient elution of 95% （V/V） acetonitrile-H20 both containing 0. 1% （V/V） formic acid and 2.0 mmol/L ammonium formate, and detected by positive electrospray ionization tandem mass spectrometry in the multiple reaction monitoring （MRM） mode,and quantified by matrix-match standard solution. Results The calibration curves were linear in the range of 30 - 10 000,50 - 10 000 and 30 - 10 000 μg/kg of tetradotoxin in muscle and liver of puffer fish and in muscle of nassarius, respectively. The correlation coefficients were within 0. 9963 - 0. 9990. The limits of detection were 10,20 and 10 μg/kg, and that of quantitation were 30,50 and 30 txg/kg for muscle and liver of puffer fish and muscle of nssarius,respectively. The average recoveries were 81.5% -93.1% ,82. 3% - 106. 0% and 83.5% - 95. 2% for tetrodotoxin spiked in muscle and liver of puffer fish and in muscle of nssarius, respectively, with relative standard deviation （RSD） of 2. 3% - 11%, 4. 3% - 14. 0% and 3.5% - 13.0% （n = 6）. Conclusion The method was simple, accurate and sensitive, and could be successfully applied to the measurement of tetrodotoxin in puffer fish and nassarius.