超高效液相色谱法快速测定烟草中的吲哚乙酸和吲哚丙酸
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摘要
本研究基于自主设计的集萃取、过滤和转移功能为一体的样品萃取瓶,建立了超高效液相色谱法(UPLC)测定烟草中的吲哚乙酸和吲哚丙酸的分析方法。采用ACQUITY UPLC BEH C18(2.1×50 mm, 1.7 m)色谱柱,流动相为30%乙腈(0.01 mol/L的乙酸水溶液),样品经80%的甲醇高速匀浆萃取、MCI-GEL CHP 20 P反向树脂净化和过滤后,通过UPLC-FL进行分析检测。结果表明:自主设计的样品萃取瓶可缩短前处理时间,吲哚乙酸在1.0～210 ng/m L、吲哚丙酸在1.0～240 ng/m L内均具有良好的线性关系(线性相关系数>0.999);吲哚乙酸、吲哚丙酸的检出限分别为0.20 ng/mL与0.25 ng/m L;在不同添加水平下,平均回收率在90.5%～103.8%之间,日内精密度和日间精密度均小于3.8%,具有较好的重复性。该方法能够满足烟草中吲哚乙酸和吲哚丙酸的快速、准确测定。
The method for the determination of indole acetic acid and indole propionic acid in tobacco by ultra-high performance liquid chromatography(UPLC) was established, based on a self-designed sample extraction bottle which combines extraction, filtration and transfer functions. The ACQUITY UPLC BEH C18(2.1×50 mm,1.7 m) chromatographic column was used, and the mobile phase was 30% acetonitrile(0.01 mol/L acetic acid aqueous solution). Samples were extracted by 80% methanol high speed homogenate, then purified and filtered by MCI-GEL CHP 20 P, analyzed and determined by UPLC-FL. The results showed that the self-designed sample extraction bottle could shorten the pretreatment time, with good linearity(R>0.999), in the range of 1.0～210 ng/mL of indole acetic acid and 1.0～240 ng/m L of indole propionic acid, the detection limits of indole acetic acid and indole propionic acid were 0.20 ng/mL and 0.25 ng/mL, respectively. Under the different levels of addition, the average recovery was between 90.5% and 103.8%. The intraday precision and the daily inter-assay precision were less than3.8% and had good repeatability. The method could satisfy the rapid and accurate determination of indole acetic acid and indole propionic acid in tobacco.
The method for the determination of indole acetic acid and indole propionic acid in tobacco by ultra-high performance liquid chromatography(UPLC) was established, based on a self-designed sample extraction bottle which combines extraction, filtration and transfer functions. The ACQUITY UPLC BEH C18(2.1×50 mm,1.7 m) chromatographic column was used, and the mobile phase was 30% acetonitrile(0.01 mol/L acetic acid aqueous solution). Samples were extracted by 80% methanol high speed homogenate, then purified and filtered by MCI-GEL CHP 20 P, analyzed and determined by UPLC-FL. The results showed that the self-designed sample extraction bottle could shorten the pretreatment time, with good linearity(R>0.999), in the range of 1.0～210 ng/mL of indole acetic acid and 1.0～240 ng/m L of indole propionic acid, the detection limits of indole acetic acid and indole propionic acid were 0.20 ng/mL and 0.25 ng/mL, respectively. Under the different levels of addition, the average recovery was between 90.5% and 103.8%. The intraday precision and the daily inter-assay precision were less than3.8% and had good repeatability. The method could satisfy the rapid and accurate determination of indole acetic acid and indole propionic acid in tobacco.
引文
Fu J.H.,Chu J.F.,Wang J.D.,and Yan C.Y.,2009,Determination of auxin in Arabidopsis Thaliana by solid ph ase extraction and high performance liquid chromatography with fluorescence detection,Huaxue Fenxi(Chinese Journal of Analytical Chemistry),37(9):1324-1327(符继红,褚金芳,王吉德,闫存玉,2009,固相萃取反相高效液相色谱荧光检测法测定拟南芥中的生长素,分析化学,37(9):1324-1327)
Fu J.H.,Sun X.H.,Wang J.D.,Chu J.F.,and Yan C.Y.,2010,Progress in quantitative analysis of plant hormones,Kexue Tongbao(Chinese Science Bulletin),55(33):3163-3176(符继红,孙晓红,王吉德,褚金芳,闫存玉,2010,植物激素定量分析方法研究进展,科学通报,55(33):3163-3176)
Gao Y.,Yang L.Y.,and Ma Y.H.,2014,HPLC determination of9 flavones in rose,Lihua Jianyan(Chemical Analysis),50(3):353-356(高燕,杨丽英,马银海,2014,高效液相色谱法测定玫瑰花中9种黄酮,理化检验,50(3):353-356)
Gong X.C.,Song C.F.,Wang M.H.,Zheng F.C.,Miu W.G.,and Wang J.S.,2012,Determination of auxin content in tobacco and cotton by high performance liquid chromatography,Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Sciences),28(1):225-227(龚晓崇,宋从凤,王鸣华,郑服丛,缪卫国,王金生,2012,高效液相色谱法测定烟草和棉花中生长素含量,江苏农业学报,28(1):225-227)
Hu S.Y.,Zhang X.,Liu W.,Cui Z.W.,Kong W.S.,and Chen Y.K.,2010,Study on the simultaneous determination of sodium,potassium,calcium and magnesium in cigarette paper by ion chromatography and sample preparation with high-speed homogenization,Yunnan Daxue Xuebao(Journal of Y-unnan University),32(S):107-109(胡守毅,张霞,刘巍,崔柱文,孔维松,陈永宽,2010,匀浆法提取-离子色谱法测定卷烟纸中钾,钠,钙,镁,云南大学学报,32(S):107-109)
Lu Q.M.,Zhang W.M.,Huang C.H.,Lu M.H.,Li J.R.,and Zhang L.,2015,Analysis of auxins by solidification of floating organic drop combined with high-performance liquid chromatography,Fuzhou Daxue Xuebao(Journal of Fuzhou University),43(4):554-559(卢巧梅,张文敏,黄川辉,卢明华,励建荣,张兰,2015,悬浮固化液相微萃取-高效液相色谱法分析生长素,福州大学学报,43(4):554-559)
Xing L.Z.,and Wang B.,2017,Recent progress on the physicochemical analysis methods of plant hormones,Lvse Keji(Journal of Green Science and Technology),(17):92-94(邢露智,王蓓,2017,植物激素理化分析方法研究进展,绿色科技,(17):92-94)
Xu Y.R.,Xue J.J.,He B.,Jin Y.C.,Yang G.Y.,and Hu Q.F.,2010,HPLC determination of polyphenols in tobacco after extraction by high-speed homogenization,Lihua Jianyan(Chemical Analysis),46(4):351-353,356(徐祎然,薛景娇,贺宾,金永灿,杨光宇,胡秋芬,2010,高速匀浆提取-高效液相色谱法测定烟草样品中多酚,理化检验,46(4):351-353,356)
Zhang X.,Liu Z.H.,Yang G.Y.,Yang L.,Duan Y.X.,Liu C.B.,Chen Y.K.,and Miu M.M.,2014,Determination of organic acids in tobacco by solid phase extraction and gas chromatogarphy,Fenxi Ceshi Xuebao(Journal of Instrumental Analysis),33(5):545-550(张霞,刘志华,杨光宇,杨柳,段沅杏,刘春波,陈永宽,缪明明,2014,固相萃取富集/气相色谱法测定烟草中的9种有机酸,分析测试学报,33(5):545-550)
Fu J.H.,Sun X.H.,Wang J.D.,Chu J.F.,and Yan C.Y.,2010,Progress in quantitative analysis of plant hormones,Kexue Tongbao(Chinese Science Bulletin),55(33):3163-3176(符继红,孙晓红,王吉德,褚金芳,闫存玉,2010,植物激素定量分析方法研究进展,科学通报,55(33):3163-3176)
Gao Y.,Yang L.Y.,and Ma Y.H.,2014,HPLC determination of9 flavones in rose,Lihua Jianyan(Chemical Analysis),50(3):353-356(高燕,杨丽英,马银海,2014,高效液相色谱法测定玫瑰花中9种黄酮,理化检验,50(3):353-356)
Gong X.C.,Song C.F.,Wang M.H.,Zheng F.C.,Miu W.G.,and Wang J.S.,2012,Determination of auxin content in tobacco and cotton by high performance liquid chromatography,Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Sciences),28(1):225-227(龚晓崇,宋从凤,王鸣华,郑服丛,缪卫国,王金生,2012,高效液相色谱法测定烟草和棉花中生长素含量,江苏农业学报,28(1):225-227)
Hu S.Y.,Zhang X.,Liu W.,Cui Z.W.,Kong W.S.,and Chen Y.K.,2010,Study on the simultaneous determination of sodium,potassium,calcium and magnesium in cigarette paper by ion chromatography and sample preparation with high-speed homogenization,Yunnan Daxue Xuebao(Journal of Y-unnan University),32(S):107-109(胡守毅,张霞,刘巍,崔柱文,孔维松,陈永宽,2010,匀浆法提取-离子色谱法测定卷烟纸中钾,钠,钙,镁,云南大学学报,32(S):107-109)
Lu Q.M.,Zhang W.M.,Huang C.H.,Lu M.H.,Li J.R.,and Zhang L.,2015,Analysis of auxins by solidification of floating organic drop combined with high-performance liquid chromatography,Fuzhou Daxue Xuebao(Journal of Fuzhou University),43(4):554-559(卢巧梅,张文敏,黄川辉,卢明华,励建荣,张兰,2015,悬浮固化液相微萃取-高效液相色谱法分析生长素,福州大学学报,43(4):554-559)
Xing L.Z.,and Wang B.,2017,Recent progress on the physicochemical analysis methods of plant hormones,Lvse Keji(Journal of Green Science and Technology),(17):92-94(邢露智,王蓓,2017,植物激素理化分析方法研究进展,绿色科技,(17):92-94)
Xu Y.R.,Xue J.J.,He B.,Jin Y.C.,Yang G.Y.,and Hu Q.F.,2010,HPLC determination of polyphenols in tobacco after extraction by high-speed homogenization,Lihua Jianyan(Chemical Analysis),46(4):351-353,356(徐祎然,薛景娇,贺宾,金永灿,杨光宇,胡秋芬,2010,高速匀浆提取-高效液相色谱法测定烟草样品中多酚,理化检验,46(4):351-353,356)
Zhang X.,Liu Z.H.,Yang G.Y.,Yang L.,Duan Y.X.,Liu C.B.,Chen Y.K.,and Miu M.M.,2014,Determination of organic acids in tobacco by solid phase extraction and gas chromatogarphy,Fenxi Ceshi Xuebao(Journal of Instrumental Analysis),33(5):545-550(张霞,刘志华,杨光宇,杨柳,段沅杏,刘春波,陈永宽,缪明明,2014,固相萃取富集/气相色谱法测定烟草中的9种有机酸,分析测试学报,33(5):545-550)