同位素稀释-超高效液相色谱-串联质谱法测定中药材中的17种真菌毒素
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摘要
称取2.000g试样于50 mL离心管中,加入乙腈-水-甲酸(70+29+1)混合溶液20.0mL,浸泡60min,于振荡混匀器上提取30min,超声提取30min,离心后,取上清液2.0mL,加入同位素内标混合溶液50μL,用磷酸盐缓冲溶液稀释至20.0mL得样品溶液。样品溶液以1～3mL·min~(-1)流量通过免疫亲和柱,用5mL水淋洗免疫亲和柱,弃去全部流出液,再用2mL甲醇-乙酸(98+2)溶液洗脱免疫亲和柱2次,收集全部洗脱液于试管中,于50℃下氮吹至近干,加入乙腈(1+9)溶液0.5mL溶解残渣,离心后,上清液供超高效液相色谱-串联质谱法分析。为了校正样品净化过程和离子化过程的损失以及消除基质效应,选用稳定性同位素[13 C]为内标物。17种真菌毒素的质量浓度在一定范围内与其峰面积与内标的峰面积的比值呈线性关系,检出限(3S/N)在0.1～2.0μg·L~(-1)之间。对空白中药材样品进行加标回收试验,回收率在84.3%～104%之间,测定值的相对标准偏差(n=6)在1.9%～13%之间。
The sample(2.000 g)was placed in 50 mL centrifuge tube,and 20.0 mL of the mixed solution of acetonitrile-water-formic acid(70+29+1)was aded.The mixture was soaked for 60 min.The mixture was extracted on an oscillating mixer for 30 min and ultrasonically extracted for 30 min.After centrifugation,2.0 mL of the supernatant was taken and 50μL of the mixed solution with internal standard of isotope was added,and the mixture was diluted with phosphate buffer solution to 20.0 mL to obtain the sample solution.The sample solution was passed through the immune affinity column at a flow rate of 1-3 mL·min-1.The immune affinity column was eluted with 5 mL of water,and all effluent was discarded.Then the immune affinity column was eluted with 2 mL of methanol-acetic acid(98+2)solution for 2 times,and all the eluent was collected in the test tube.The eluent was evaporated to near-dryness by N2-blowing at 50 ℃,and 0.5 mL of acetonitrile-water(1+9)solution was used to dissolved the residue.After centrifugation,the supernatant was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry.Stable isotopes 13 C internal standards were used to correct the loss of sample purification and ionization process and eliminate matrix effect.Linear relationships between the ratios of peak areas of 17 mycotoxins to peak areas of the internal standards and the mass concentration of 17 mycotoxins were found in definite ranges,with detection limits(3 S/N)in the range of 0.1-2.0μg·L~(-1).Test for recovery was made by standard addition method on the base of blank Chinece herbal medicine sample,giving results in the range of 84.3%-104%,and RSDs(n=6)ranged from 1.9% to 13%.
The sample(2.000 g)was placed in 50 mL centrifuge tube,and 20.0 mL of the mixed solution of acetonitrile-water-formic acid(70+29+1)was aded.The mixture was soaked for 60 min.The mixture was extracted on an oscillating mixer for 30 min and ultrasonically extracted for 30 min.After centrifugation,2.0 mL of the supernatant was taken and 50μL of the mixed solution with internal standard of isotope was added,and the mixture was diluted with phosphate buffer solution to 20.0 mL to obtain the sample solution.The sample solution was passed through the immune affinity column at a flow rate of 1-3 mL·min-1.The immune affinity column was eluted with 5 mL of water,and all effluent was discarded.Then the immune affinity column was eluted with 2 mL of methanol-acetic acid(98+2)solution for 2 times,and all the eluent was collected in the test tube.The eluent was evaporated to near-dryness by N2-blowing at 50 ℃,and 0.5 mL of acetonitrile-water(1+9)solution was used to dissolved the residue.After centrifugation,the supernatant was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry.Stable isotopes 13 C internal standards were used to correct the loss of sample purification and ionization process and eliminate matrix effect.Linear relationships between the ratios of peak areas of 17 mycotoxins to peak areas of the internal standards and the mass concentration of 17 mycotoxins were found in definite ranges,with detection limits(3 S/N)in the range of 0.1-2.0μg·L~(-1).Test for recovery was made by standard addition method on the base of blank Chinece herbal medicine sample,giving results in the range of 84.3%-104%,and RSDs(n=6)ranged from 1.9% to 13%.
引文
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[4]杨美华.药用植物及其产品中真菌及真菌毒素污染研究进展[J].贵州农业科学,2008,36(6):59-63.
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[6]GB/T 8381.4-2005配合饲料中T-2毒素的测定薄层色谱法[S].
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[8]SN/T 1958-2007进出口食品中伏马毒素B1残留量检测方法酶联免疫吸附法[S].
[9]张爱婷,石延榜,张振凌,等.ELISA法测定部分种子和果实类中药黄曲霉毒素B1含量[J].医学研究杂志,2008,37(10):48~49.
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[11]张雪辉,陈建民.免疫亲和柱净化HPLC柱后溴衍生化方法检测中药中黄曲霉毒素[J].中国中药杂志,2005,30(3):182-184.
[12]韦日伟,杨小丽,仇峰,等.免疫亲和柱净化-在线柱后衍生-HPLC-FLD同时测定甘草中黄曲霉毒素B1、B2、G1、G2和赭曲霉毒素A的含量[J].中国中药杂志,2011,36(17):2342-2346.
[13]RAHMANI A,SELAMAT J,SOLEIMANY F.Optimization and validation of a HPLC method for simultaneous determination of aflatoxin B1,B2,G1,G2,ochratoxin A and zearalenone using an experimental design[J].Food Additives and Contaminants,2011,28(7):902-912.
[14]FERREIRA I,FEMANDES J O,CUNHA S C.Optimazation and validation of a method based in a QuEChERS procedure and gas chromatography-mass spectrometry for the determination of multi-mycotoxins in popcorn[J].Food Control,2012,27:188-193.
[15]郑润生,徐晖,彭苑霞,等.稀释法结合LC-MS/MS检测在10种中药材污染黄曲霉毒素高通量筛查中的应用研究[J].中国中药杂志,2014,39(2):273-277.
[16]赵孔祥,葛宝坤,陈旭艳,等.在线免疫亲和净化-液相色谱-串联质谱快速测定中草药及中成药中10种真菌毒素[J].分析化学,2011,39(9):1341-1346.
[17]探井,郑润生,王文丽,等.中药饮片中黄曲霉毒素和玉米赤霉烯酮的液质联用检测分析[J].时珍国医国药,2012,23(10):2469-2472.
[18]陈勇,陈重均,李劲,等.超高效液相色谱串联质谱法检测三七药材中10种真菌毒素[J].药学学报,2015,50(1):81-85.
[19]KIM D H,HONG S Y,KANG J W,et al.Simultaneous determination of multi-mycotoxins in cereal grains collected from south korea by LC/MS/MS[J].Toxins,2017,9(3):106-106.
[2]黄莉,张浩,丁伟琴,等.中药中真菌毒素污染问题[J].海峡药学,2009,21(6):95-99.
[3]张成,豆小文,杨美华.中药中常见真菌毒素毒理及快速检测技术[J].中国药理学与毒理学杂志,2016,30(12):1369-1378.
[4]杨美华.药用植物及其产品中真菌及真菌毒素污染研究进展[J].贵州农业科学,2008,36(6):59-63.
[5]李俊媛,万丽,杨美华.真菌毒素限量标准及其在中药中的研究进展[J].中草药,2011,42(3):602-609.
[6]GB/T 8381.4-2005配合饲料中T-2毒素的测定薄层色谱法[S].
[7]GB/T 8381.6-2005配合饲料中脱氧雪腐镰刀菌烯醇的测定薄层色谱法[S].
[8]SN/T 1958-2007进出口食品中伏马毒素B1残留量检测方法酶联免疫吸附法[S].
[9]张爱婷,石延榜,张振凌,等.ELISA法测定部分种子和果实类中药黄曲霉毒素B1含量[J].医学研究杂志,2008,37(10):48~49.
[10]ARNAUD H,FLORIAN C,CHRISTOPHE D.Effect of sampling and extraction on deoxynivalenol quantification[J].Food Chemistry,2011,127(1):303-307.
[11]张雪辉,陈建民.免疫亲和柱净化HPLC柱后溴衍生化方法检测中药中黄曲霉毒素[J].中国中药杂志,2005,30(3):182-184.
[12]韦日伟,杨小丽,仇峰,等.免疫亲和柱净化-在线柱后衍生-HPLC-FLD同时测定甘草中黄曲霉毒素B1、B2、G1、G2和赭曲霉毒素A的含量[J].中国中药杂志,2011,36(17):2342-2346.
[13]RAHMANI A,SELAMAT J,SOLEIMANY F.Optimization and validation of a HPLC method for simultaneous determination of aflatoxin B1,B2,G1,G2,ochratoxin A and zearalenone using an experimental design[J].Food Additives and Contaminants,2011,28(7):902-912.
[14]FERREIRA I,FEMANDES J O,CUNHA S C.Optimazation and validation of a method based in a QuEChERS procedure and gas chromatography-mass spectrometry for the determination of multi-mycotoxins in popcorn[J].Food Control,2012,27:188-193.
[15]郑润生,徐晖,彭苑霞,等.稀释法结合LC-MS/MS检测在10种中药材污染黄曲霉毒素高通量筛查中的应用研究[J].中国中药杂志,2014,39(2):273-277.
[16]赵孔祥,葛宝坤,陈旭艳,等.在线免疫亲和净化-液相色谱-串联质谱快速测定中草药及中成药中10种真菌毒素[J].分析化学,2011,39(9):1341-1346.
[17]探井,郑润生,王文丽,等.中药饮片中黄曲霉毒素和玉米赤霉烯酮的液质联用检测分析[J].时珍国医国药,2012,23(10):2469-2472.
[18]陈勇,陈重均,李劲,等.超高效液相色谱串联质谱法检测三七药材中10种真菌毒素[J].药学学报,2015,50(1):81-85.
[19]KIM D H,HONG S Y,KANG J W,et al.Simultaneous determination of multi-mycotoxins in cereal grains collected from south korea by LC/MS/MS[J].Toxins,2017,9(3):106-106.