配合飼料中三種鐮刀菌毒素檢測(cè)方法的研究

1、配合飼料中三種鐮刀菌毒素檢測(cè)方法的研究摘 要本試驗(yàn)通過(guò)優(yōu)化提取方法、衍生處理?xiàng)l件和儀器條件，分別建立了氣相色譜-質(zhì)譜聯(lián)用、液相色譜-質(zhì)譜聯(lián)用檢測(cè)配合飼料中脫氧雪腐鐮刀菌烯醇(DON)和雪腐鐮刀菌烯醇(NIV)的方法，以及液相色譜-質(zhì)譜聯(lián)用檢測(cè)配合飼料中玉米赤霉烯酮(ZAE)的方法。配合飼料中 DON 和 NIV 提取采用乙腈-水(84:16,V/V)為提取溶劑，溶劑量為100ml，振蕩提取 60min。Mycosep227 多功能柱做為凈化柱。氣相色譜-質(zhì)譜聯(lián)用同時(shí)檢測(cè) DON 和 NIV，衍生處理采用七氟丁酰咪唑 70衍生 50min，或者 TMSI-BSA-TMCS(3:3:2,V/V/V

2、)室溫衍生 15min。HP-5 MS 毛細(xì)管柱分離，電子轟擊離子源電離，質(zhì)譜檢測(cè)。SCAN 模式全掃描，特征離子做定性分析，提取離子做定量分析。氟化衍生 DON和 NIV 的提取離子為 294、333，硅烷化衍生提取離子為 422、379。加標(biāo)量在20700ng/g 的范圍內(nèi)，氟化衍生氣相色譜-質(zhì)譜檢測(cè)方法中 DON 和 NIV 的回收率分別為85.591.7%、74.883.1%，檢出限為 3ng/g，RSD9.0%。硅烷化衍生氣相色譜-質(zhì)譜檢測(cè)方法中 DON 和 NIV 的回收率分別為 81.792.6%、73.886.6%，檢出限為6ng/g、8ng/g，RSD4.9。氟化衍生檢測(cè)靈敏

3、度高，但衍生過(guò)程需加熱、耗時(shí)，衍生物不及硅烷化衍生物穩(wěn)定。液相色譜-質(zhì)譜同時(shí)檢測(cè) DON 和 NIV，色譜柱為 Agilent XDB-C18 柱，甲醇-0.1乙酸銨水溶液(20:80,V/V)為流動(dòng)相，大氣壓化學(xué)電離后質(zhì)譜檢測(cè)，檢測(cè)離子為負(fù)離子。信號(hào) 1SCAN 模式掃描，特征離子做定性分析，信號(hào) 2 SIM 選擇離子模式掃描做定量分析，DON 和 NIV 的選擇離子為 295、311。加標(biāo)量 20700ng/g 的范圍內(nèi)，DON 和 NIV 的回收率分別為 91.1103.6%，85.692.7%，檢出限分別為 15ng/g，18ng/g，RSD5.9%。與氣相色譜-質(zhì)譜聯(lián)用相比，該方法不

4、需衍生，易于操作，但檢測(cè)靈敏度不及前者。配合飼料中 ZEA 提取采用乙腈-水(75:25,V/V)為提取溶劑，溶劑量為 150ml，攪拌提取 40min，Mycosep226 多功能柱作為凈化柱。液相色譜-質(zhì)譜檢測(cè)飼料中玉米赤霉烯酮，Agilent XDB-C18 柱為色譜柱，甲醇-0.1乙酸銨水溶液(75:25,V/V)為流動(dòng)相，大氣壓化學(xué)電離后質(zhì)譜檢測(cè)，檢測(cè)離子為正離子。信號(hào) 1 SCAN 模式掃描，特征離子做定性分析，信號(hào) 2 SIM 選擇離子模式掃描做定量分析，ZEA 的選擇離子為 319。加標(biāo)量20700ng/g 的范圍內(nèi)，ZEA 的回收率為 89.3101.7%，檢出限為 1ng/

5、g。RSD7.3%。本研究將氣相色譜-質(zhì)譜對(duì)真菌毒素的檢測(cè)范圍擴(kuò)展，建立同時(shí)檢測(cè) DON 和 NIV 的方法，并在國(guó)內(nèi)首次將液相色譜-質(zhì)譜聯(lián)用方法運(yùn)用到鐮刀菌毒素的檢測(cè)中。以上建立的三種方法與國(guó)標(biāo)法相比，獲得了更高的靈敏度和重現(xiàn)性，減少了有毒溶劑的使用，操作步驟簡(jiǎn)單。以上方法有望成為谷物中鐮刀菌毒素檢測(cè)的主要方法。關(guān)鍵詞：配合飼料；氣相色譜-質(zhì)譜；液相色譜-質(zhì)譜；脫氧雪腐鐮刀菌烯醇；雪腐鐮刀菌烯醇；玉米赤霉烯酮THE STUDY OF THE METHODS FOR THE DETERMINATION OF THREE MAJOR FUSARIUM MYCOTOXINS IN FORMULA

6、FEEDABSTRACTGC-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

7、by Mycosep227.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 i

8、ons 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 flu

9、oroacylation is 3ng/g, RSD9.0%. The recoveries of DON and NIV added to feed at the range of 20700ng/g are 85.591.7% and 74.883.1% 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

10、 added to feed at the range of 20700ng/g are 81.792.6% and 73.886.6% respectively, RSD4.9%.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 metho

11、d 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, Sign

12、al 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 91.1103.6% for DON and 85.692.7% for NIV added to the feed at the range of 20700ng/

13、g . The detection limit is 15ng/g for DON and 18ng/g for NIV, RSD5.9%.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 neednt derivating in this method, the procedure of this method is simpler.T

14、he 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 Mycosep226. Agilent XDB-C18 is used as column; methanol-0.1% ammonium acetate solution (75:25, V/V) is used as mobile phase. The to

15、xins 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 20700ng/g are 89.

16、3101.7%. The detection limit is 1ng/g for ZEA, RSD7.3%.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 bette