UPLC-MS/MS法与HPLC-FLD法分析检测中药中的黄曲霉毒素
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摘要
目的:建立黄曲霉毒素G_2、G_1、B_2、B_1的超高效液相色谱串联三重四极杆质谱法(UPLC-MS/MS法),同时采用高效液相色谱荧光法(HPLC-FLD法)进行测定。方法:采用UPLC-MS/MS测定,待测黄曲霉毒素的中药经过免疫亲和柱净化,采用C18(2.1 mm×50 mm,1.7μm)进行色谱分离,柱温30℃,以含0.1%甲酸的2.0 mmol·L~(-1)乙酸铵溶液(A)-甲醇(B)为流动相,梯度洗脱,流速0.3 mL·min~(-1),用电喷雾正离子多反应监测模式(MRM)扫描;采用HPLC联用柱后衍生荧光检测方法测定同样的样品;并验证检测结果。结果:UPLC-MS/MS黄曲霉毒素质谱检测的线性关系良好,定量下限范围在0.177 8～0.633 3μg·kg~(-1),检测下限范围在0.055 1～0.190 0μg·kg~(-1),样品测定结果在1.423～1.975μg·kg~(-1)之间;同时采用HPLC-FLD荧光检测法进行测定(定量下限范围为0.660～0.204μg·kg~(-1),检测下限范围为0.059～0.206μg·kg~(-1))。同一样品测定结果误差在1.501%～9.377%之间。结论:与柱后衍生化HPLC-FLD法相比,液质联用测定中药材中黄曲霉毒素的方法,专属性强,操作简单,简便快速,灵敏度高,减少实验成本。
Objective:To establish a method for the determination of aflatoxins in Chinese materia medica by an ultra-high performance liquid chromatography with triple quadrupole mass spectrometry(UPLC-MS/MS)and to conduct simultaneous determination by HPLC with fluorescence detection(HPLC-FLD).Methods:The herbal extract was purified by immune affinity column and separated by a C_1,(2.1 mm × 50 mm,1.7 μm)column(temperature:30 ℃)and the mobile phase consisted of 2.0 mmol·L~(-1) ammonium acetate-0.1%formic water-methanol at a flow rate of 0.3 mL·min~(-1).Multiple-reaction monitoring(MRM)was performed to identify and quantify four aflatoxins(G_2,G_1,B_2,B_1).In addition,the HPLC coupled with the post-column derivatization fluorescence detection method was conducted as a validated analysis.Results:Good linearity was obtained in UPLC-MS/MS method.The limit of quantification(LOQ)ranged from 0.177 8-0.633 3 μg·kg~(-1) and the limit of detection(LOD)ranged from 0.055 1-0.190 0 μg·kg~(-1).The determination results were between 1.423 and 1.975 μg·kg~(-1).Meanwhile,samples were determined by the fluorescence detection method(LOQ,0.660-0.204μg·kg~(-1) LOD,0.059-0.206 μg·kg~(-1)),and the relative errors of results obtained by the two methods were 1.501%-9.377%.Conclusion:Compared with the HPLC-FLD coupled with the post-column derivatization method,the UPLC-MS/MS method is specific,simple,fast and sensitive,which can reduce the experimentalcost.
Objective:To establish a method for the determination of aflatoxins in Chinese materia medica by an ultra-high performance liquid chromatography with triple quadrupole mass spectrometry(UPLC-MS/MS)and to conduct simultaneous determination by HPLC with fluorescence detection(HPLC-FLD).Methods:The herbal extract was purified by immune affinity column and separated by a C_1,(2.1 mm × 50 mm,1.7 μm)column(temperature:30 ℃)and the mobile phase consisted of 2.0 mmol·L~(-1) ammonium acetate-0.1%formic water-methanol at a flow rate of 0.3 mL·min~(-1).Multiple-reaction monitoring(MRM)was performed to identify and quantify four aflatoxins(G_2,G_1,B_2,B_1).In addition,the HPLC coupled with the post-column derivatization fluorescence detection method was conducted as a validated analysis.Results:Good linearity was obtained in UPLC-MS/MS method.The limit of quantification(LOQ)ranged from 0.177 8-0.633 3 μg·kg~(-1) and the limit of detection(LOD)ranged from 0.055 1-0.190 0 μg·kg~(-1).The determination results were between 1.423 and 1.975 μg·kg~(-1).Meanwhile,samples were determined by the fluorescence detection method(LOQ,0.660-0.204μg·kg~(-1) LOD,0.059-0.206 μg·kg~(-1)),and the relative errors of results obtained by the two methods were 1.501%-9.377%.Conclusion:Compared with the HPLC-FLD coupled with the post-column derivatization method,the UPLC-MS/MS method is specific,simple,fast and sensitive,which can reduce the experimentalcost.
引文
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[5]刘书宇,仇峰,杨美华.免疫亲和柱净化-在线柱后光化学衍生HPLC-FLD检测莲子中黄曲霉毒素B-1,B-2,G-1,G-2及其液质确证[J].中国中药杂志,2012,37(3):305LIU SY,QIU F,YANG MH.Determination of aflatoxins in Nelumbinis Semen by immunoaffinity column clean-up and HPLCFLD with on-line post-column photochemical derivatization and LC-MS/MS confirmation[J].China J Chin Mater Med,2012,37(3):305
[6]计融,柳祯,江涛,等.总黄曲霉毒素ELISA定量检测试剂盒研制[J].中国公共卫生,2007,23(3):331JI R,LIU Z,JIANG T,et al.Development of ELISA-kit of quantitative analysis for aflatoxins[J].Chin J Public Health,2007,23(3):331
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[8]曹纪亮,孔维军,杨美华,等.真菌毒素快速检测方法研究进展[J].药物分析杂志,2013,33(1):159CAO JL,KONG WJ,YANG MH,et al.Research progress on rapid detection methods of mycotoxins[J].Chin J Pharm Anal,2013,33(1):159
[9]励炯,唐敏,王红青,等.HPLC-MS/MS法测定灵芝孢子粉黄曲霉毒素含量[J].中药材,2017,40(9):2060LI J,TANG M,WANG HQ,et al. Detection of four aflatoxins in Ganoderma lucidum spore powder by HPLC-MS/MS[J].J Chin Med Mater,2017,40(9):2060
[10]ZHANG Z,HU X,ZHANG Q,et al. Determination for multiple mycotoxins in agricultural products using HPLC-MS/MS via a multiple antibody immunoaffinity column[J].J Chromatogr B,2016,1021:145
[11]SIDDIQUE NA,MUJEEB M,AHMAD S,et al. Determination of aflatoxins in medicinal plants by high-performance liquid chromatography-tandem mass spectrometry[J].J Pharm Pharm Sci,2012,16(2):321
[12]SUN M,XU D,WANG S,et al. Inkjet-based dispersive liquid-liquid microextraction method coupled with UHPLC-MS/MS for the determination of aflatoxins in wheat[J].Anal Chem,2019,91(4):3027
[13]PARK J,KIM DH,MOON JY,et al. Distribution analysis of twelve mycotoxins in corn and corn-derived products by LC-MS/MS to evaluate the carry-over ratio during wet-milling[J].Toxins(Basel),2018,10(8):319
[14]SHEKHAR M,SINGH N,DUTTA R,et al. Comparative study of qualitative and quantitative methods to determine toxicity level of Aspergillus flavus isolates in maize[J].PLoS One,2017,12(12):e0189760
[15]YAMASAKI T,MIYAKE S,SATO N,et al. Development of enzyme-linked immunosorbent assay for analysis of total aflatoxins based on monoclonal antibody reactive with aflatoxins B1,B2,G1 and G2[J].Shokuhin Eiseigaku Zasshi,2018,59(5):200
[16]HORWITZ W.Official Methods of Analysis of the Association of Official Analytical Chemists[M].17th Ed.Arlington:Association of Official Analytical Chemists,2005:2(chapter 49)
[2]IARC Working Group on the Evaluation of Carcinogenic Risks to Humans.Some Naturally Occurring Substances:Food Items and Constituents,Heterocyclic Aromatic Amines,and Mycotoxins[M].Lyon:World Health Organization,International Agency for Research on Cancer,1993
[3]中华人民共和国药典2015年版.一部[S].2015:224ChP 2015.Vol I[S].2015:224
[4]苏小洁,潘显茂.用免疫亲和柱净化-柱后衍生高效液相色谱荧光法检测山楂干中多种黄曲霉毒素含量的效果观察[J].当代医药论丛,2018,16(16):189SU XJ,PAN XM.Determination of multiple aflatoxins in dried hawthorn by immunoaffinity column clean-up and HPLC-FLD with post-column derivatization[J].Contemp Med Symp,2018,16(16):189
[5]刘书宇,仇峰,杨美华.免疫亲和柱净化-在线柱后光化学衍生HPLC-FLD检测莲子中黄曲霉毒素B-1,B-2,G-1,G-2及其液质确证[J].中国中药杂志,2012,37(3):305LIU SY,QIU F,YANG MH.Determination of aflatoxins in Nelumbinis Semen by immunoaffinity column clean-up and HPLCFLD with on-line post-column photochemical derivatization and LC-MS/MS confirmation[J].China J Chin Mater Med,2012,37(3):305
[6]计融,柳祯,江涛,等.总黄曲霉毒素ELISA定量检测试剂盒研制[J].中国公共卫生,2007,23(3):331JI R,LIU Z,JIANG T,et al.Development of ELISA-kit of quantitative analysis for aflatoxins[J].Chin J Public Health,2007,23(3):331
[7]RIZZO I,VEDOYA G,MAURUTTO S,et al. Assessment of toxigenic fungi on Argentinean medicinal herbs[J].Microbiol Res,2004,159(2):113
[8]曹纪亮,孔维军,杨美华,等.真菌毒素快速检测方法研究进展[J].药物分析杂志,2013,33(1):159CAO JL,KONG WJ,YANG MH,et al.Research progress on rapid detection methods of mycotoxins[J].Chin J Pharm Anal,2013,33(1):159
[9]励炯,唐敏,王红青,等.HPLC-MS/MS法测定灵芝孢子粉黄曲霉毒素含量[J].中药材,2017,40(9):2060LI J,TANG M,WANG HQ,et al. Detection of four aflatoxins in Ganoderma lucidum spore powder by HPLC-MS/MS[J].J Chin Med Mater,2017,40(9):2060
[10]ZHANG Z,HU X,ZHANG Q,et al. Determination for multiple mycotoxins in agricultural products using HPLC-MS/MS via a multiple antibody immunoaffinity column[J].J Chromatogr B,2016,1021:145
[11]SIDDIQUE NA,MUJEEB M,AHMAD S,et al. Determination of aflatoxins in medicinal plants by high-performance liquid chromatography-tandem mass spectrometry[J].J Pharm Pharm Sci,2012,16(2):321
[12]SUN M,XU D,WANG S,et al. Inkjet-based dispersive liquid-liquid microextraction method coupled with UHPLC-MS/MS for the determination of aflatoxins in wheat[J].Anal Chem,2019,91(4):3027
[13]PARK J,KIM DH,MOON JY,et al. Distribution analysis of twelve mycotoxins in corn and corn-derived products by LC-MS/MS to evaluate the carry-over ratio during wet-milling[J].Toxins(Basel),2018,10(8):319
[14]SHEKHAR M,SINGH N,DUTTA R,et al. Comparative study of qualitative and quantitative methods to determine toxicity level of Aspergillus flavus isolates in maize[J].PLoS One,2017,12(12):e0189760
[15]YAMASAKI T,MIYAKE S,SATO N,et al. Development of enzyme-linked immunosorbent assay for analysis of total aflatoxins based on monoclonal antibody reactive with aflatoxins B1,B2,G1 and G2[J].Shokuhin Eiseigaku Zasshi,2018,59(5):200
[16]HORWITZ W.Official Methods of Analysis of the Association of Official Analytical Chemists[M].17th Ed.Arlington:Association of Official Analytical Chemists,2005:2(chapter 49)
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