体外稳定同位素标记-液相色谱-串联质谱法测定谷物中4种霉菌毒素的含量  

Determination of 4 Mycotoxins in Grains by Liquid Chromatography-Tandem Mass Spectrometry with Stable Isotope Labeling in Vitro

作　　者：吴铨欣 孙良娟 余文华 吴欣蕾 张雯静 丁秀琼 张娜 李红权 郭宁 WU Quanxin;SUN Liangjuan;YU Wenhua;WU Xinlei;ZHANG Wenjing;DING Xiuqiong;ZHANG Na;LI Hongquan;GUO Ning(School of Pharmacy,Guangdong Medical University,Dongguan 523808,China;Zhanjiang Customs Technology Center,Zhanjiang 524022,China)

机构地区：[1]广东医科大学药学院,东莞523808 [2]湛江海关技术中心,湛江524022

出　　处：《理化检验(化学分册)》2025年第1期1-9,共9页Physical Testing and Chemical Analysis(Part B:Chemical Analysis)

基　　金：广东省中医药局科研项目(20212102);广东省医学科研基金:项目(A2021121);海关总署科研项目(2021HK205);湛江市创新创业团队引育“领航计划”(2020LHJH005);国家自然科学基金:(81803723);广东省湛江市科技计划项目(2020B01021);广东医科大学大学生创新创业训练计划项目(GDMU2020109)。

摘　　要：提出了体外稳定同位素标记-液相色谱-串联质谱法(LC-MS/MS)测定谷物中黄曲霉毒素B_(1)(AFB_(1))、黄曲霉毒素B_(2)(AFB_(2))、黄曲霉毒素M_(1)(AFM_(1))、玉米赤霉烯酮(ZEN)等4种霉菌毒素含量的方法。取过40目(0.425 mm)筛的样品200 mg置于离心管中,加入0.5 mL甲醇,涡旋1 min,离心3 min,共提取3次,合并上清液,用氮气吹干,加入170μL甲醇溶解,再依次加入20μL吉拉德P(GP)试剂(20 mmol·L^(-1))和10μL冰乙酸,涡旋10 s,于50℃振荡3 h,用氮气吹干,加入190μL甲醇溶解,再加入10μL GP-d_(5)标记的霉菌毒素标准品(内标)溶液(2.0μmol·L^(-1)),混匀,采用LC-MS/MS测定其中4种霉菌毒素的含量。以ODS-3 C_(18)色谱柱为固定相,以不同体积比的0.1%(体积分数)甲酸溶液-乙腈混合液为流动相进行梯度洗脱,质谱分析采用电喷雾离子(ESI)源,在正、负离子扫描模式下以多反应监测(MRM)模式检测。结果表明,4种霉菌毒素的浓度在一定范围内与对应的峰面积和内标峰面积比呈线性关系,检出限(3S/N)为24~150 pmol·L^(-1)。按照标准加入法进行回收试验,回收率为79.2%~119%,日内精密度与日间精密度试验所得测定值的相对标准偏差(n=9)均不大于20%。方法用于实际60份谷物样品的分析,部分样品检出AFB_(1)、AFB_(2)、AFM_(1)、ZEN,检出量分别为0.757~1.981μg·kg^(-1),0.060~1.059μg·kg^(-1),0.313~0.538μg·kg^(-1),0.969~4.348μg·kg^(-1)。A method for determination of 4 mycotoxins,including aflatoxin B_(1)(AFB_(1)),aflatoxin B_(2)(AFB_(2)),aflatoxin M_(1)(AFM_(1)),and zeralenone(ZEN)in grains by liquid chromatography-tandem mass spectrometry(LC-MS/MS)with stable isotope labeling in vitro was proposed.The sample(200 mg)that passed through a 40 mesh(0.425 mm)sieve was taken and placed in a centrifuge tube.0.5 mL of methanol was added,and the mixture was vortexed for 1 min and centrifuged for 3 min.The extraction process was repeated 3 times,and the supernatant was combined and blown to dryness by nitrogen.170μL of methanol was added to dissolve,and 20μL of Girard P(GP)reagent(20 mmol·L^(-1))and 10μL of glacial acetic acid were added sequentially.The solution was vortexed for 10 s and shaken for 3 h at 50℃,and blown to dryness by nitrogen.190μL of methanol was added to dissolve,and 10μL of GP-d_(5) labeled mycotoxins standard(internal standard)solution(2.0μmol·L^(-1))was added.The 4mycotoxins in the mixed solution were determined by LC-MS/MS.ODS-3 C_(18)chromatographic column was used as the stationary phase and the mixed solution composed of 0.1%(volume fraction)formic acid solution and acetonitrile at different volume ratios was used as the mobile phase for gradient elution.Electrospray ion(ESI)source was used for mass spectrometry.The targets were detected by multiple reaction monitoring(MRM)mode in positive and negative ion scanning mode.As shown by the results,linear relationships between the ratios of the corresponding peak area to the internal standard peak area and mass concentrations of4 mycotoxins were found in definite ranges,with detection limits(3S/N)in the range of 24-150 pmol·L^(-1).Test for recovery was made by the standard addition method,giving results in the range of 79.2%-119%.Tests for precision of intra-day and interday were made,with RSDs(n=9)of determined values not more than 20%.This method was used for the analysis of 60 actual grain samples,and AFB_(1),AFB_(2),AFM_(1),and ZEN were detected,with detection amounts

关 键 词：体外稳定同位素标记 液相色谱-串联质谱法 谷物 霉菌毒素 

分 类 号：O657.63[理学—分析化学]