化妆品生产工艺论文和食品中的微生物检测论文

配合饲料中三种镰刀菌毒素检测方法的研究 摘 要 本试验通过优化提取方法、衍生处理条件和仪器条件，分别建立了气相色谱-质谱联用、液相色谱-质谱联用检测配合饲料中脱氧雪腐镰刀菌烯醇(DON)和雪腐镰刀菌烯醇(NIV)的方法，以及液相色谱-质谱联用检测配合饲料中玉米赤霉烯酮(ZAE)的方法。 配合饲料中DON和NIV提取采用乙腈-水(84:16,V/V)为提取溶剂，溶剂量为100ml，振荡提取60min。Mycosep®227多功能柱做为净化柱。气相色谱-质谱联用同时检测DON和NIV，衍生处理采用七氟丁酰咪唑70℃衍生50min，或者TMSI-BSA-TMCS(3:3:2,V/V/V)室温衍生15min。HP-5 MS毛细管柱分离，电子轰击离子源电离，质谱检测。SCAN模式全扫描，特征离子做定性分析，提取离子做定量分析。氟化衍生DON和NIV的提取离子为294、333，硅烷化衍生提取离子为422、379。加标量在20～700ng/g的范围内，氟化衍生气相色谱-质谱检测方法中DON和NIV的回收率分别为85.5～91.7%、74.8～83.1%，检出限为3ng/g，RSD≤9.0%。硅烷化衍生气相色谱-质谱检测方法中DON和NIV的回收率分别为81.7～92.6%、73.8～86.6%，检出限为6ng/g、8ng/g，RSD≤4.9％。氟化衍生检测灵敏度高，但衍生过程需加热、耗时，衍生物不及硅烷化衍生物稳定。 液相色谱-质谱同时检测DON和NIV，色谱柱为Agilent XDB-C18柱，甲醇-0.1％乙酸铵水溶液(20:80,V/V)为流动相，大气压化学电离后质谱检测，检测离子为负离子。信号1SCAN模式扫描，特征离子做定性分析，信号2 SIM选择离子模式扫描做定量分析，DON和NIV的选择离子为295、311。加标量20～700ng/g的范围内，DON和NIV的回收率分别为91.1～103.6%，85.6～92.7%，检出限分别为15ng/g，18ng/g，RSD≤5.9%。与气相色谱-质谱联用相比，该方法不需衍生，易于操作，但检测灵敏度不及前者。 配合饲料中ZEA提取采用乙腈-水(75:25,V/V)为提取溶剂，溶剂量为150ml，搅拌提取40min，Mycosep®226多功能柱作为净化柱。液相色谱-质谱检测饲料中玉米赤霉烯酮，Agilent XDB-C18柱为色谱柱，甲醇-0.1％乙酸铵水溶液(75:25,V/V)为流动相，大气压化学电离后质谱检测，检测离子为正离子。信号1 SCAN模式扫描，特征离子做定性分析，信号2 SIM选择离子模式扫描做定量分析，ZEA的选择离子为319。加标量20～700ng/g的范围内，ZEA的回收率为89.3～101.7%，检出限为1ng/g。RSD≤7.3%。 本研究将气相色谱-质谱对真菌毒素的检测范围扩展，建立同时检测DON和NIV的方法，并在国内首次将液相色谱-质谱联用方法运用到镰刀菌毒素的检测中。以上建立的三种方法与国标法相比，获得了更高的灵敏度和重现性，减少了有毒溶剂的使用，操作步骤简单。以上方法有望成为谷物中镰刀菌毒素检测的主要方法。 关键词：配合饲料；气相色谱-质谱；液相色谱-质谱；脱氧雪腐镰刀菌烯醇；雪腐镰刀菌烯醇；玉米赤霉烯酮 THE STUDY OF THE METHODS FOR THE DETERMINATION OF THREE MAJOR FUSARIUM MYCOTOXINS IN FORMULA FEED ABSTRACT GC-MS and HPLC-MS were studied and applied to determinate DON, NIV and ZEA in formula feed. The conditions of extraction, derivation and instrument have been optimized. The DON and NIV in feed are extracted with 100ml acetonitrile-water (84:16, V/V), vibrating for 60 min, then cleaned-up by Mycosep®227.The method of determination of DON and NIV by GC-MS is used HFBI as derivatization reagent, the toxins are derivatived at 70℃ for 50min, or used TMSI-BSA-TMCS (3:3:2, V/V/V) as derivatization reagent, derivatived at room temperature for 15min. After seperated by HP-5 MS columnSpecific ions are used for qualitative analysis, extracted ions for quantitative analysis. The extracted ion of fluoroacylation for DON is 294 and for NIV is 333, the extracted ions of trimethylsilylation for DON and NIV are 422 and 379 respectively. The detection limit of the method of determination after fluoroacylation is 3ng/g, RSD≤9.0%. The recoveries of DON and NIV added to feed at the range of 20～700ng/g are 85.5～91.7% and 74.8～83.1% respectively. The detection limit of the method of determination after trimethylsilylation for DON and NIV are 6ng/g and 8ng/g respectively. The recoveries of DON and NIV added to feed at the range of 20～700ng/g are 81.7～92.6% and 73.8～86.6% respectively, RSD≤4.9%.It shows good precision of the method of determination after fluoroacylation, but the fluoroacylation need heating and spending longer time, the HFB derivatives are more instable than TMS derivatives. The method of determination of DON and NIV by HPLC-MS uses Agilent XDB-C18 as column, methanol-0.1％ ammonium acetate solution(20:80,V/V) is used as mobile phase, the toxins ionized by APCI, are determinated by MS using negative ions. Signal 1 is SCAN mode, specific ions are used for qualitative analysis, Signal 2 is SIM mode, selected ions are used for quantitative analysis, and the selective ions for DON and NIV are 295 and 311 respectively. The recoveries of the method for the determination of DON and NIV by HPLC-MS are 91.1～103.6% for DON and 85.6～92.7% for NIV added to the feed at the range of 20～700ng/g . The detection limit is 15ng/g for DON and 18ng/g for NIV, RSD≤5.9%. The sensitivity of the method of determination of DON and NIV by HPLC-MS is lower than the method of determination of DON and NIV by GC-MS, but the toxins needn’t derivating in this method, the procedure of this method is simpler. The method of determination of ZEA by HPLC-MS is as follows: The feed is extracted with 150 ml acetonilie-water (75:25, V/V), whisking for 40 min, and then cleaned-up by Mycosep®226. Agilent XDB-C18 is used as column; methanol-0.1% ammonium acetate solution (75:25, V/V) is used as mobile phase. The toxins ionized by APCI, are determinated by MS using positive ions. Specific ions are used for qualitative analysis, selected ion is used for quantitative analysis, and the selective ion for ZEA is 319. The recoveries of the method of determination of ZEA added to feed at the range of 20～700ng/g are 89.3～101.7%. The detection limit is 1ng/g for ZEA, RSD≤7.3%. The study enlarges the scope of determination of mycotoxins by GC-MS, and introduces HPLC-MS to determinate Fusarium mycotoxins for the first time domestically. Compared with the national standard methods, the three methods mentioned above shows lower detection limits and better reproductivity. Besides these, the methods use less poisonous solvent and the operational procedures are simpler. They are hopeful to be the major methods of determination of fusarium mycotoxins in corn. KEY WOEDS: formula feed；GC-MS；HPLC-MS；DON；NIV；ZEA