果品、蔬菜中赤霉素GA_3残留量的测定
摘要
赤霉素是一大类植物激素，属于双萜类，现已分离鉴定的有100余种赤霉素。其中，尤以赤霉酸最普遍、最重要。它是一种广普性植物生长调节剂，几乎在植物生长、发育的各个阶段都起着调节作用，尤其对蔬菜、水果等有着显著的增产作用。作为大环内酯抗生素，美国、日本等规定水果蔬菜中赤霉素最高残留限量为0.2ppm。我国赤霉素残留分析一般采用荧光检测技术，其样品前处理常用液液萃取、薄层分离、柱层层析法。这些方法操作繁琐，且需使用大量有机溶剂。本课题利用固相萃取(SPE)前处理方法，建立了多种高灵敏度、快速的方法对果品、蔬菜中痕量赤霉素残留物进行了检测，并将它们予以比较，为确定赤霉素残留标准做基础研究。
第一，采用固相萃取前处理方法净化样品，考察了样品在C_(18)上的保留行为，纯化效果显著。实验用HP-1色谱柱，无需柱前衍生化，直接进样气相色谱一质谱(GC-MS)分析。以选择离子作为质谱监测目标化合物的方式，可有效排除干扰，提高检测灵敏度1～2个数量级，使得果品中赤霉素残留的分析更为简便可行。
第二，建立了固相萃取-高效液相色谱(SPE-HPLC)法分析番茄中赤霉素GA_3残留量的方法。研究了不同填料的固相萃取小柱对番茄中赤霉素的萃取回收率的影响，以及各自的纯化结果。通过比较：用C_(18)小柱净化，可以得到满足要求的样品净化液。优化条件下C_(18)小柱萃取样品效果最好，固相萃取起到了好的净化效果，特别是对样品提取液中色素、可溶性糖、酸、酯的去除。该方法的线性范围为0.21～1.05×10~2mg/L，检测限为0.011mg/Kg，加标回收率范围88.45％～95.90％，其RSD为1.15％～6.56％。
第三，采用以电喷雾电离(ESI)为接口的液相色谱质谱(HPLC-MS)联用技术，选择ESI负离子测定模式，对赤霉素GA_3进行了质谱分析；并建立了番茄中赤霉素残留的定量测定方法。与传统的液相色谱-紫外检测分析方
馨
硕士学位论文
MAS’I’卜R’S‘1’l!ESIS
法相比较，液相色谱一质谱方法可以排出背景杂质的干扰，具有更高的选择性
和灵敏度，可简化样品的提取步骤。实验中对电喷雾参数进行了优化，采用多
离子反应监测(MRM)方式进行定量测定时，方法的检测限为0.02呵Kg。样
品加标回收率为%.2%一104.6%，精密度为4.63%一6.95%。该方法尤其适于复
杂的多组分混合物中痕量物质的测定，是一种测定植物中赤霉素残留量的好方
法。
第四，参照中华人民共和国进出口商品检验行业标准SN 035卜95，.采用固
相萃取净化样品，利用赤霉素乙醇溶液与浓硫酸配位成一种对光有吸收并能发
射比较强辐射的荧光黄基体，从而辐射荧光的原理，建立了荧光光谱法测定草
荀、橙子中赤霉素GA3残留量。实验中优化了分析条件，该法灵敏度好，但专
一性稍差，经SPE纯化后的部分杂质对测定GA3荧光值影响较大，测定加标
回收结果不及色谱测定法。
Gibberellins are a big kind of phytohormone and they are of biterpene. At preset, people have separated and determined more than one hundred kinds of Gibberellins. Among them, gibberellic acid(GA3)is the most popular and important one. It is a kind of eurytopic growth hormone, regulating the plant' s upgrowth and development at its every step. Specially, it has obvious production-increacing effect on vegetables and fruits. As a kind of big-ring lactone antibiotic, it is prescribed that
the maximum residue limitation of gibberellin is less than 0.2ppm in the USA and
Japan. Usually in China we use the fluorescence technology for the analysis of the
gibberellin residue. The pre-disposal of the sample is often adopted liquid-liquid extraction, the separation of thin-layer chromatography and the column chromatography. However the operation of these methods is cockamamie and needs a great amount of organic solvent. In this thesis the author set up the high-sensitivity and fast methods to detect the gibberellic acid residue in the vegetables and fruits by the purification of solid phase extraction. Also the methods has been given comparation between each other, which make out the basic reseach for the confirmation of the detecting standard of gibberellin residue.
In the first place, a method of solid phase extraction followed by gas chromatography-mass spectrometry with selected ion mode for the direct determination of gibberellin residue in fruit samples has been developed. The retention behaviors of gibberellin samples on C18 small column were investigated. With the use of HP-1 capillary column and selected ion mode of MS, the disturbance from various origin can be overcomed. Its sensitivity is higher than full scan detection method, which makes the detection of gibberellin residue easier and faster.
In the second place, with solid phase extraction, a method of high performance
liquid chromatography for the determination of gibberellin residue in tomato has been developed. The effection of different kinds of filling on gibberellin recovery in samples and their purified results are investigated. By comparison, we know that we can gain the satisfied refined sample solution with the purification of C18 small column. Under the optimized conditions, the purified effection of C18 small column is best, especially in the removement of the coloring matter, the soluble sugar and ester in the sample extraction. The linear range of this method is 0.20mg/L~l .05 X 102mg/L. Its detection limit is 0.011 mg/Kg, the average recoveries are 88.45%~ 95.90% and the RSD is from 1.15% to 6.56%.
In the third place, by using the coupled technology of liquid-mass and the selection of the negative ion mode of electric spray ion, we have make a mass analysis about gibberellin and set up its residue quantified metod in tomato samples. Compared with the traditional liquid chromatography with UV detection, the LC-MS can remove the disturbance of the background impurity. It has higher selection and sensitivity and can predigest the extrction step of the sample. In the experiment we have optimized the ESI parameters. The detection limits of this method is 0.02ng/Kg, the average recoveries are 96.2%-104.6% and the RSD is from 4.63 % to 6.95 %. The method is very suited for the the determination of the trace substance in the complex multiple mixture, it is a good method for the detection of gibberellin residue in the plant.
In the forth place, referring to the imports and exports commodinity trade testing standard of People's Republic of ChinaCSN 0350-95), based on the principle that, corporating with sulfuric acid, the alcohol solution of gibberellin will obtain a kind of fluorescence huang's matrix which can absorb rays and eradiate stronger radiation. With the purification of solid phase extraction, a fluorescence spectra analysis method for the determination of the gibberellin residues in the orange and strawberry has been developed by testing the fluorescence intensity of the fluorescence huang's matrix; In the experiment we optimize the ana
第一，采用固相萃取前处理方法净化样品，考察了样品在C_(18)上的保留行为，纯化效果显著。实验用HP-1色谱柱，无需柱前衍生化，直接进样气相色谱一质谱(GC-MS)分析。以选择离子作为质谱监测目标化合物的方式，可有效排除干扰，提高检测灵敏度1～2个数量级，使得果品中赤霉素残留的分析更为简便可行。
第二，建立了固相萃取-高效液相色谱(SPE-HPLC)法分析番茄中赤霉素GA_3残留量的方法。研究了不同填料的固相萃取小柱对番茄中赤霉素的萃取回收率的影响，以及各自的纯化结果。通过比较：用C_(18)小柱净化，可以得到满足要求的样品净化液。优化条件下C_(18)小柱萃取样品效果最好，固相萃取起到了好的净化效果，特别是对样品提取液中色素、可溶性糖、酸、酯的去除。该方法的线性范围为0.21～1.05×10~2mg/L，检测限为0.011mg/Kg，加标回收率范围88.45％～95.90％，其RSD为1.15％～6.56％。
第三，采用以电喷雾电离(ESI)为接口的液相色谱质谱(HPLC-MS)联用技术，选择ESI负离子测定模式，对赤霉素GA_3进行了质谱分析；并建立了番茄中赤霉素残留的定量测定方法。与传统的液相色谱-紫外检测分析方
馨
硕士学位论文
MAS’I’卜R’S‘1’l!ESIS
法相比较，液相色谱一质谱方法可以排出背景杂质的干扰，具有更高的选择性
和灵敏度，可简化样品的提取步骤。实验中对电喷雾参数进行了优化，采用多
离子反应监测(MRM)方式进行定量测定时，方法的检测限为0.02呵Kg。样
品加标回收率为%.2%一104.6%，精密度为4.63%一6.95%。该方法尤其适于复
杂的多组分混合物中痕量物质的测定，是一种测定植物中赤霉素残留量的好方
法。
第四，参照中华人民共和国进出口商品检验行业标准SN 035卜95，.采用固
相萃取净化样品，利用赤霉素乙醇溶液与浓硫酸配位成一种对光有吸收并能发
射比较强辐射的荧光黄基体，从而辐射荧光的原理，建立了荧光光谱法测定草
荀、橙子中赤霉素GA3残留量。实验中优化了分析条件，该法灵敏度好，但专
一性稍差，经SPE纯化后的部分杂质对测定GA3荧光值影响较大，测定加标
回收结果不及色谱测定法。
Gibberellins are a big kind of phytohormone and they are of biterpene. At preset, people have separated and determined more than one hundred kinds of Gibberellins. Among them, gibberellic acid(GA3)is the most popular and important one. It is a kind of eurytopic growth hormone, regulating the plant' s upgrowth and development at its every step. Specially, it has obvious production-increacing effect on vegetables and fruits. As a kind of big-ring lactone antibiotic, it is prescribed that
the maximum residue limitation of gibberellin is less than 0.2ppm in the USA and
Japan. Usually in China we use the fluorescence technology for the analysis of the
gibberellin residue. The pre-disposal of the sample is often adopted liquid-liquid extraction, the separation of thin-layer chromatography and the column chromatography. However the operation of these methods is cockamamie and needs a great amount of organic solvent. In this thesis the author set up the high-sensitivity and fast methods to detect the gibberellic acid residue in the vegetables and fruits by the purification of solid phase extraction. Also the methods has been given comparation between each other, which make out the basic reseach for the confirmation of the detecting standard of gibberellin residue.
In the first place, a method of solid phase extraction followed by gas chromatography-mass spectrometry with selected ion mode for the direct determination of gibberellin residue in fruit samples has been developed. The retention behaviors of gibberellin samples on C18 small column were investigated. With the use of HP-1 capillary column and selected ion mode of MS, the disturbance from various origin can be overcomed. Its sensitivity is higher than full scan detection method, which makes the detection of gibberellin residue easier and faster.
In the second place, with solid phase extraction, a method of high performance
liquid chromatography for the determination of gibberellin residue in tomato has been developed. The effection of different kinds of filling on gibberellin recovery in samples and their purified results are investigated. By comparison, we know that we can gain the satisfied refined sample solution with the purification of C18 small column. Under the optimized conditions, the purified effection of C18 small column is best, especially in the removement of the coloring matter, the soluble sugar and ester in the sample extraction. The linear range of this method is 0.20mg/L~l .05 X 102mg/L. Its detection limit is 0.011 mg/Kg, the average recoveries are 88.45%~ 95.90% and the RSD is from 1.15% to 6.56%.
In the third place, by using the coupled technology of liquid-mass and the selection of the negative ion mode of electric spray ion, we have make a mass analysis about gibberellin and set up its residue quantified metod in tomato samples. Compared with the traditional liquid chromatography with UV detection, the LC-MS can remove the disturbance of the background impurity. It has higher selection and sensitivity and can predigest the extrction step of the sample. In the experiment we have optimized the ESI parameters. The detection limits of this method is 0.02ng/Kg, the average recoveries are 96.2%-104.6% and the RSD is from 4.63 % to 6.95 %. The method is very suited for the the determination of the trace substance in the complex multiple mixture, it is a good method for the detection of gibberellin residue in the plant.
In the forth place, referring to the imports and exports commodinity trade testing standard of People's Republic of ChinaCSN 0350-95), based on the principle that, corporating with sulfuric acid, the alcohol solution of gibberellin will obtain a kind of fluorescence huang's matrix which can absorb rays and eradiate stronger radiation. With the purification of solid phase extraction, a fluorescence spectra analysis method for the determination of the gibberellin residues in the orange and strawberry has been developed by testing the fluorescence intensity of the fluorescence huang's matrix; In the experiment we optimize the ana
引文
[1]罗平．饮料分析与检验，中国轻工业出版社，1993
[2]王运浩等．食品农药残留与分析控制技术展望，现代科学仪器，2003，No．1，8-13
[3]Van Zoonen P, Yan der Hoff G R. LC-GC, 1998, 16(1): 21-25
[4]Majors R E, Raynie D E. LC-GC, 1997, 15(12): 1106-1115
[5]张燕婉．色谱法用于食品分析时的样品预处理，食品研究和开发，1995，16(2)：42-44
[6]王平华．现代分析技术在食品科研应用中的新进展，农业工程学报，1995，11(2)，138-142
[7]樊德方．农药残留量分析与检测，上海科学技术出版社，1982
[8]汪正范等．色质联用技术，化学工业出版社，2001
[9]Bercello D, Hennion M C. Anal. Chim. Acta, 1995, 318(1-3): 1-41
[10]Pichon V, Hennion M C. Anal. Chim. Acta, 1998, 376(1): 21-35
[11]Larcorte S, Barcelo D. Anal. Chem., 1996, 68:2464-2470
[12]Aguillar C, Ferrer I, Borrull F, et al. J. Chromatogra., 1996, A754:77-84
[13]夏锦尧．实用荧光分析法，公安大学出版社，1991年
[14]李泰华，陈卉．原子与分子物理学报，1995，Vol．12(2)：143-148
[2]王运浩等．食品农药残留与分析控制技术展望，现代科学仪器，2003，No．1，8-13
[3]Van Zoonen P, Yan der Hoff G R. LC-GC, 1998, 16(1): 21-25
[4]Majors R E, Raynie D E. LC-GC, 1997, 15(12): 1106-1115
[5]张燕婉．色谱法用于食品分析时的样品预处理，食品研究和开发，1995，16(2)：42-44
[6]王平华．现代分析技术在食品科研应用中的新进展，农业工程学报，1995，11(2)，138-142
[7]樊德方．农药残留量分析与检测，上海科学技术出版社，1982
[8]汪正范等．色质联用技术，化学工业出版社，2001
[9]Bercello D, Hennion M C. Anal. Chim. Acta, 1995, 318(1-3): 1-41
[10]Pichon V, Hennion M C. Anal. Chim. Acta, 1998, 376(1): 21-35
[11]Larcorte S, Barcelo D. Anal. Chem., 1996, 68:2464-2470
[12]Aguillar C, Ferrer I, Borrull F, et al. J. Chromatogra., 1996, A754:77-84
[13]夏锦尧．实用荧光分析法，公安大学出版社，1991年
[14]李泰华，陈卉．原子与分子物理学报，1995，Vol．12(2)：143-148