中药材农药残留快速分析新策略：基于保留指数的八角茴香中12个菊酯类农药GC-QQQ-MS/MS测定方法的建立

目的：探索建立一种在八角茴香复杂基质中利用科瓦茨保留指数辅助定性的12个拟除虫菊酯类农药化合物GC-QQQ-MS/MS定量方法。方法：采用三重四极杆气质联用仪，SHIMADZU SH-Rxi-5Sil MS毛细管柱（30 m×0.25 mm×0.25 μm）；进样口温度250℃，进样量1.0 μL，不分流进样，高压进样压力为250 kPa；载气为高纯氦气，载气控制方式为恒线速度模式；色谱柱流量1.69 mL·min^-1，线速度47.2 cm·s^-1，吹扫流量为5 mL·min^-1；程序升温：初始温度50℃，保持1 min，先以25℃·min^-1升温至125℃，再以10℃·min^-1升温至300℃，保持15 min；柱平衡时间为2 min。离子源为电子轰击源，离子化能量70 eV，离子源温度230℃，质谱传输接口温度250℃，碰撞气为氩气；质谱监测模式为多反应监测，溶剂延迟时间为6 min。利用正构烷烃C9~C33对照品的保留时间结合SmartDatabase_Pesticides数据库，计算出12个目标农药化合物的预测保留时间，用于快速筛查样品中存在的农药和辅助定性。以氘代倍硫磷为内标物质，制备12个目标农药化合物的标准曲线，以内标标准曲线法计算样品中目标农药化合物的含量。结果：目标农药化合物（包括同分异构体）的预测保留时间和实测保留时间非常接近，时间差均小于0.02 min，证明了保留指数在中药材复杂基质溶液中辅助定性的准确性和可行性。测定结果显示，20批次八角茴香样品中有2个批次检测到了微量的氯氰菊酯，1个批次检测到了微量的氯菊酯。结论：基于保留指数辅助定性的八角茴香中12个拟除虫菊酯类农药化合物的GC-QQQ-MS/MS定量方法具有较好的适用性，对其他中药材多农药残留检测方法的建立具有参考价值。

Objective:To establish a GC-QQQ-MS/MS quantitative method for the determination of 12 pyrethroid pesticides in complex basis of Anisi Stellati Fructus based on Kovats retention index. Methods:This study was performed on a GC-QQQ-MS/MS spectrometer,equipped with a SHIMADZU SH-Rxi-5Sil MS column(30 m×0.25 mm×0.25 μm). The temperature of the injection port was 250℃,the injection volume was 1.0 μL,the injection mode was splitless,the injection pressure was 250 kPa;the carrier gas was high purity helium and the carrier gas control mode was constant linear velocity mode;the column flow rate was 1.69 mL·min^-1,the line speed was 47.2 cm·s^-1,the purge flow rate was 5 mL·min^-1. The temperature rise method was the gradient program:the initial temperature was 50℃,kept for 1 minute,first raised the temperature to 125℃ at a speed of 25℃ per minute,and then raised the temperature to 300℃ at a speed of 10℃ per minute,kept for 15 minutes;the balance time was 2 minutes. The ion source was an electron bombardment source,the ionization energy was 70 eV,the ion source temperature was 230℃,the mass spectrometry transmission interface temperature was 250℃,the collision gas was argon gas,and the mass spectrometry monitoring mode was multiple reaction monitoring,the solvent delay time was 6 min. Using retention times of n-alkanes C9-C33 combined with the smartDatabase_Pesticides database,the predicted retention times of the 12 target pesticides were calculated. The predicted retention times could be used to quickly screen out the pesticides existed in the sample and to help identify the target pesticideds. Using fenthion-d₆ as the internal reference substance,the standard curves of 12 targeted pesticides were prepared. The contents of the targeted pesticides in the sample were calculated by the internal standard curve method. Results:The predicted retention times of 25 targeted pesticides(including isomers)were very close to the actual measured retention times with deviations below 0.02 min,which proved the accuracy and feasibility of the retention index in qualitative analysis for complex basis of traditional Chinese medicine. Quantitative determination of 12 pesticides in 20 batches of Anisi Stellati Fructus samples were carried out. The results showed that trace cypermethrin was detected in 2 batches of samples with contents ranging from 15.22 μg·kg^-1(YP201718)to 38.19 μg·kg^-1(YP201703). Permethrin was detected in 1 batch of sample,and the content was 24.05 μg·kg^-1. Conclusion:The newly developed GC-QQQ-MS/MS method based on retention index-assisted qualitative for quantitative analysis of 12 pyrethroid pesticides in Anisi Stellati Fructus is feasible,and this method has reference value for establishment of multi-pesticides residue assay method for other Chinese medicinal materials.

[1] 人民文摘.中药材农药残留的秘密[R/OL].2013[2018-04-10].http://paper.people.com.cn/rmwz/html/2013-09/01/content_1307472.htm. People digest.Pesticide Residues in Herbal Medicines-Investigation Report on Pesticide Pollution of Herbal Medicines[R/OL].2013[2018-04-10].http://paper.people.com.cn/rmwz/html/2013-09/01/content_1307472.htm.
[2] 傅巧真,林慧彬,林建强,等.中药材农药残留原因及防治措施的研究进展[J].时珍国医国药,2014,25(4):925 FU QZ,LIN HB,LIN JQ,et al.Research progress of pesticide residue in Chinese herbal medicine causes and prevention measures[J].Lishizhen Med Mater Med Res,2014,25(4):925
[3] 王丽丽,夏会龙.我国中草药中农药残留的特点[J].中草药,2007,38(3):471 WANG LL,XIA HL.Characteristics of pesticide residues in Chinese herbal medicines in China[J].Chin Tradit Herb Drugs,2007,38(3):471
[4] 宁艳.拟除虫菊酯生殖毒性及中药干预作用研究[D].广州:广州中医药大学,2006:16 NING Y.The Study of Chinese Herba1 Medicine to Interfere Toxicity on Reproduction Correlating with Pyrethroid[D].Guangzhou:Guangzhou University of Traditional Chinese Medicine,2006:16
[5] 冯坚.具有光学活性的拟除虫菊酯杀虫剂开发概况[J].农药,2000,39(2):1 FENG J.Development of optically active pyrethroid insecticides[J].Pesticides,2000,39(2):1
[6] 塔娜,李想,孙成,等.拟除虫菊酯类农药的非对映和对映异构体分离[J].中国环境科学,2006,26(1):87 TA N,LI X,SUN C,et al.Diastereomeric and enantiomeric separation of pyrethroid pesticides[J].Chin Environ Sci,2006,26(1):87
[7] 李朝阳,张智超,张玲,等.拟除虫菊酯农药手性分离的研究进展[J].现代农药,2006,5(5):1 LI CY,ZHANG ZC,ZHANG L,et al.Research progress on chiral separation of pyrethroid pesticides[J].Mod Agrochem,2006,5(5):1
[8] 刘开孔,祥虹,李莹,等.GC-MS/MS法测定8种植物提取物中15种拟除虫菊酯类农药的残留量[J].分析试验室,2014,33(8):930 LIU KK,XIANG H,LI Y,et al.Determination of 15 pyrethroid pesticide residues in 8 plant extracts by GC-MS/MS[J].Chin J Anal Lab,2014,33(8):930
[9] 刘永波,贾立,张明霞,等.GC-MS法测定拟除虫菊酯类农药残留[J].理化检验(化学分册),2006,42(8):637 LIU YB,JIA L,ZHANG MX,et al.Determination of pyrethroid pesticide residues by GC-MS[J].Phys Test Chem Anal(Part B:Chem Anal),2006,42(8):637
[10] 刘旭,钟怀宁,杨雪梅,等.川芎等9种药材中拟除虫菊酯类农药残留的检测[J].时珍国医国药,2006,17(2):154 LIU X,ZHONG HN,YANG XM,et al.Determination of the residues of cypermethrin and fenvalerate in nine medical materials[J].Lishizhen Med Mater Med Res,2006,17(2):154
[11] 高天兵,张曙明,田金改.三七、白芍及西洋参中氯氰菊酯、氰戊菊酯和溴氰菊酯的残留量测定[J].药物分析杂志,1999,19(5):313 GAO TB,ZHANG SM,TIAN JG.Determination of residual amount of cypermethrin,fenvalerate and deltamethrin in Chinese herbal medicines by capillary gas chromatography(CGC-ECD)[J].Chin J Pharm Anal,1999,19(5):313
[12] 赵梦瑶,王旗,杨秀伟.吴茱萸中拟除虫菊酯类农药残留分析[J].药物分析杂志,2007,27(9):1344 ZHAO MY,WANG Q,YANG XW.Determination of pyrethroids pesticide residues in Fructus Evodiae as traditional Chinese medicine[J].Chin J Pharm Anal,2007,27(9):1344
[13] KOVATS,E.Gas-chromatographische Charakterisierung organischer Verbindungen.Teil 1:retentionsindices aliphatischer halogenide,alkohole,aldehyde und ketone[J].Helv Chim Acta,1958,41(7):1915
[14] ZHANG J,FANG AQ,WANG B,et al.A retention index tool for analysis of gas chromatography-mass spectrometry data[J].J Chromatogr A,2011,1218(37):6522
[15] 赵晨曦,梁逸曾,胡黔楠,等.气相色谱保留指数定性方法研究进展[J].分析化学,2005,33(5):715 ZHAO CX,LIANG YZ,HU QN,et al.Research progress on qualitative methods of gas chromatography retention index[J].Chin J Anal Chem,2005,33(5):715
[16] KOO I,SHI X,KIM S,et al.Compound identification using retention index for analysis of gas chromatography-mass spectrometry data[J].J Chromatogr A,2014,1337:202
[17] GARCIA-DOMINGUEZ JA,LEBRON-AGUILAR R,QUINTANILLA-LÓPEZ JE.An accurate and easy procedure to obtain isothermal Kovats retention indices in gas chromatography[J].J Sep Sci,2006,29(18):2785
[18] MIHALEVA VV,VERHOEVEN HA,de VOS RC,et al.Automated procedure for candidate compound selection in GC-MS metabolomics based on prediction of Kovats retention index[J].Bioinformatics,2009,25(6):787
[19] 中华人民共和国药典2015年版.四部[S].2015:212,224,374 ChP 2015.Vol Ⅳ[S].2015:212,224,374
[20] ICH Topic Q2B Validation of Analytical Procedures:Methodology(CPMP/ICH/281/95).Vol 4[S].1996:6
[21] 夏品华,张明时,王兴宁,等.气相色谱法测定中药材中拟除虫菊酯类农药残留量[J].中国卫生检验杂志,2008,18(2):257 XIA PH,ZHANG MS,WANG XN,et al.Simultaneous determination of residues of pyrethroid pesticides in medicinal plants by gas chromatography[J].Chin J Health Lab Technol,2008,18(2):257
[22] 王莹.中药中GC-MS农药多残留法检测平台的建立[D].北京:中国食品药品检定研究院,2011:25 WANG Y.Establishment of Detection Platform for GC-MS Pesticide Multi-residue Method in Traditional Chinese Medicine[D].Beijing:National Institutes for Food and Drug Control,2011:25
[23] 钱韵旭.国外新农残标准对中药出口的影响及对策研究[D].沈阳:沈阳药科大学,2014:36 QIAN YX.The Influence of Foreign New Rural Residue Standards on Chinese Medicine Export and Its Countermeasures[D].Shenyang:Shenyang Pharmaceutical University,2014:36