超高效液相色谱-飞行时间质谱法定性定量检测乌头类植物中14种乌头生物碱及其代谢产物的含量
|
摘要
目的建立超高效液相色谱-飞行时间质谱(ultra performance liquid chromatography-quadrupole-time of flight mass spectrometer, UPLC/Q-TOF)同时定性定量检测乌头类植物中14种乌头生物碱及其代谢产物的含量的分析方法。方法采用SPE固相萃取小柱提取净化,用超高效液相色谱串联四级杆飞行时间质谱,Dual AJS ESI源,正离子模式下采集的数据进行定性定量分析。色谱柱为Aglient SB-C_(18)(2.1 mm×50 mm,2.7μm),流动相为乙腈-0.1%甲酸水(含5 mmol/L乙酸铵),梯度洗脱,流速0.3 mL/min。结果14种乌头生物碱及其代谢产物在10～100 ng/mL范围内具有良好的线性关系,相关系数均大于0.99,检出限为0.1～5.0 ng/mL,定量限为0.5～10 ng/mL,加标回收率为81.2%～113.7%(相对标准偏差(relative standard deviation, RSD)<10%, n=6)。结论该方法能快速、准确地对乌头类植物中的乌头生物碱成分进行定性定量检测,对该植物毒性和药效的深入研究提供了一种新的方法。
Objective To establish a method for qualitative and quantitative analysis of 14 kinds of aconitine alkaloids and their metabolites in Aconitum L.by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometer(UPLC/Q-TOF).Methods The samples were extracted and purified by SPE solid phase extraction cartridge,and the data was collected by ultra-high performance liquid chromatography tandem flight time mass spectrometry with Dual AJS ESI source,and analyzed qualitatively and quantitatively in positive ion mode.Chromatographic column was Agilent SB-C_(18)(2.1 mm×50 mm,2.7?m)and the flow phase was acetonitrile-0.1%formic acid aqueous solution containing 5 mmol/L ammonium acetate(gradient elution)at a flow rate of 0.3 mL/min.Results The 14 kinds of aconitum alkaloids and their metabolites had good linear relationship in the range of10-100 ng/mL,and the correlation coefficients were greater than 0.99.The limits of detection were 0.1-5.0 ng/mL,the limits of quantification were 0.5-10 ng/mL,and the recovery rates were 81.2%-113.7%(RSD<10%,n=6).Conclusion This method can quickly and accurately determine the qualitative and quantitative detection of aconite alkaloids in aconite plants,which provides a new method for the in-depth study of the phytotoxicity and efficacy of the plant.
Objective To establish a method for qualitative and quantitative analysis of 14 kinds of aconitine alkaloids and their metabolites in Aconitum L.by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometer(UPLC/Q-TOF).Methods The samples were extracted and purified by SPE solid phase extraction cartridge,and the data was collected by ultra-high performance liquid chromatography tandem flight time mass spectrometry with Dual AJS ESI source,and analyzed qualitatively and quantitatively in positive ion mode.Chromatographic column was Agilent SB-C_(18)(2.1 mm×50 mm,2.7?m)and the flow phase was acetonitrile-0.1%formic acid aqueous solution containing 5 mmol/L ammonium acetate(gradient elution)at a flow rate of 0.3 mL/min.Results The 14 kinds of aconitum alkaloids and their metabolites had good linear relationship in the range of10-100 ng/mL,and the correlation coefficients were greater than 0.99.The limits of detection were 0.1-5.0 ng/mL,the limits of quantification were 0.5-10 ng/mL,and the recovery rates were 81.2%-113.7%(RSD<10%,n=6).Conclusion This method can quickly and accurately determine the qualitative and quantitative detection of aconite alkaloids in aconite plants,which provides a new method for the in-depth study of the phytotoxicity and efficacy of the plant.
引文
[1]尹田鹏.六种云南产乌头属植物的生物碱成分研究[D].昆明:云南大学,2016.Yin TP. Alkaloids of six Aconitum plants from Yunnan province[D].Kunming:Yunnan University, 2016.
[2]袁晓航.中药川乌与草乌的鉴别比较及药理活性探究[J].内蒙古中医药,2017, 36(18):137-138.Yuan XH. Traditional Chinese medicin(TCM)in the identification of radix aconiti and radix aconiti compare and pharmacological activitiy[J].New Mongol J Trad Chin Med, 2017, 36(18):137-138.
[3]徐辉.回阳救逆用附子[J].东方药膳,2016,(9):46.Xu H. Reviving yang for fuzi[J]. Orient Herb, 2016,(9):46.
[4]安婧娴,刘芳,刘芳,等.近年来乌头属植物二萜生物碱化学成分及其镇痛活性研究进展[J].中南药学,2016,(5):521-525.An JX, Liu F, Liu F, et al. Recent research progress on diterpenoid alkaloids from genus aconitum and their analgesic activity[J]. Centr South Pharm, 2016,(5):521-525.
[5]赵翡翠,李杰.乌头属药用植物中生物碱的研究进展[J].中国现代应用药学,2010,(S1):1177-1182.Zhao FC, Li J. Recent progress in research of alkaloid in Aconitum plants[J]. Chin J Mod Appl Pharm, 2010,(S1):1177-1182.
[6]王丹,贾德贤,李真真,等.草乌的安全性评价与风险控制措施的探讨[J].中国中药杂志,2018, 43(15):3093-3100.Wang D, Jia DX, Li ZZ, et al. Safety evaluation and risk control measLures for Aconite kusnezoffii radix[J]. Chin J Chin Mat Med, 2018, 43(15):3093-3100.
[7]黄建明,郭济贤,孙明明,等.草乌中生物碱含量测定方法的研究[J].中药材,2002, 25(12):878-880.Huang JM, Guo JX, Sun MM, et al. The method for determination of aconitum alkaloids in radix Aconiti kusnezoffii[J]. Chin Med Mat, 2002,25(12):878-880.
[8]段晓燕,沈勇.滇西乌头二萜生物碱成分研究[J].中国药师,2017,20(9):1525-1528.Duan XY, Shen Y. Diterpenoid alkaloids in the roots Aconitum bulleyanum diels[J]. Chin Pharm, 2017, 20(9):1525-1528.
[9]丁祥,侯怡铃,罗利.德钦乌头根部化学成分的研究[J].昆明理工大学学报(自然科学版),2012, 37(6):62-66.Ding X, Hou YL, Luo L. Study on chemical compositon from roots of Aconitum ouvrardianum hand-mazz[J]. J Kunming Univ Sci Technol(Nat Sci Ed),2012, 37(6):62-66.
[10]杨秀伟.生物碱[M].北京:化学工业出版社,2005.Yang XW. Alkaloid[M]. Beijing:Chemical Industry Press, 2005.
[11]张轩,周斌,路慧丽.草乌甲素的药理作用机制及临床应用研究进展[J].慢性病学杂志,2016,(11):1210-1213.Zhang X, Zhou B, Lu HL. Advances in pharmacological mechanism and clinical application of aconitine[J]. Chron Pathematol J, 2016,(11):1210-1213.
[12]邓鸣,蒋受军,朱斌.飞行时间质谱分析技术在植物药研究中的应用进展[J].华西药学杂志,2013, 28(1):12-15.Deng M, Jiang SJ, Zhu B. Progress in the application of time-of-flight mass spectrometry in the study of botanical drugs[J]. West Chin J Pharm Sci, 2013, 28(1):12-15.
[13] Jiang T, Jiang Z, Mukadaisi S, et al. Research progress in hydrolysis mechanism and LC-MS determination method of genus Aconitum L. based on traditional Chinese medicine simulation processing[J]. Chin Trad Herb Drug, 2016,(12):121-130.
[14] GA/T934-2011生物样品中次乌头碱、乌头碱、中乌头碱的液相色谱-串联质谱检验方法[S].GA/T 934-2011 Determination of sub aconitine, aconitine and meconoxine in biological samples by liquid chromatography-tandem mass spectrometry[S].
[15] Lei S, Wang SC, Sun MQ, et al. Fragmentation pathways of six aconitine alkaloids using HPLC-Q-TOF-MS technology[J]. Chin Trad Herb Drug,2016,(16):2827-2828.
[16]欧水平.基于“一体多用”的乌头新药用部位及制剂研究-川乌头茎叶治疗类风湿性关节炎的成药性研究[D].成都:成都中医药大学,2013.Ou SP. New part and preparation for medicine of aconitum carmichaeli debx. based on"A integrated plant but multi-medicine"-the druggability research of leaf and stem in treating rheumatoid arthritis[D]. Chengdu:Chengdu University of Traditional Chinese Medicine, 2013.
[17]蒋荡,侯大斌,邓文伟,等.不同产地附子中附子灵和宋果灵含量的比较[J].北方园艺,2013,(6):159-161.Jiang D, Hou DB, Deng WW, et al. Study on content comparison of fuziline and songorine from daughter roots of aconitum carmicichaeli produced from different areas[J]. Northern Horticult, 2013,(6):159-161.
[18]杨杏林,杨大男,杨清锐,等.草乌甲素的研究进展[J].世界临床药物,2013,34(2):80-83.Yang XL, Yang DN, Yang QR, et al. Research advances of bulleyaconitine A[J]. World Clin Drug, 2013, 34(2):80-83.
[2]袁晓航.中药川乌与草乌的鉴别比较及药理活性探究[J].内蒙古中医药,2017, 36(18):137-138.Yuan XH. Traditional Chinese medicin(TCM)in the identification of radix aconiti and radix aconiti compare and pharmacological activitiy[J].New Mongol J Trad Chin Med, 2017, 36(18):137-138.
[3]徐辉.回阳救逆用附子[J].东方药膳,2016,(9):46.Xu H. Reviving yang for fuzi[J]. Orient Herb, 2016,(9):46.
[4]安婧娴,刘芳,刘芳,等.近年来乌头属植物二萜生物碱化学成分及其镇痛活性研究进展[J].中南药学,2016,(5):521-525.An JX, Liu F, Liu F, et al. Recent research progress on diterpenoid alkaloids from genus aconitum and their analgesic activity[J]. Centr South Pharm, 2016,(5):521-525.
[5]赵翡翠,李杰.乌头属药用植物中生物碱的研究进展[J].中国现代应用药学,2010,(S1):1177-1182.Zhao FC, Li J. Recent progress in research of alkaloid in Aconitum plants[J]. Chin J Mod Appl Pharm, 2010,(S1):1177-1182.
[6]王丹,贾德贤,李真真,等.草乌的安全性评价与风险控制措施的探讨[J].中国中药杂志,2018, 43(15):3093-3100.Wang D, Jia DX, Li ZZ, et al. Safety evaluation and risk control measLures for Aconite kusnezoffii radix[J]. Chin J Chin Mat Med, 2018, 43(15):3093-3100.
[7]黄建明,郭济贤,孙明明,等.草乌中生物碱含量测定方法的研究[J].中药材,2002, 25(12):878-880.Huang JM, Guo JX, Sun MM, et al. The method for determination of aconitum alkaloids in radix Aconiti kusnezoffii[J]. Chin Med Mat, 2002,25(12):878-880.
[8]段晓燕,沈勇.滇西乌头二萜生物碱成分研究[J].中国药师,2017,20(9):1525-1528.Duan XY, Shen Y. Diterpenoid alkaloids in the roots Aconitum bulleyanum diels[J]. Chin Pharm, 2017, 20(9):1525-1528.
[9]丁祥,侯怡铃,罗利.德钦乌头根部化学成分的研究[J].昆明理工大学学报(自然科学版),2012, 37(6):62-66.Ding X, Hou YL, Luo L. Study on chemical compositon from roots of Aconitum ouvrardianum hand-mazz[J]. J Kunming Univ Sci Technol(Nat Sci Ed),2012, 37(6):62-66.
[10]杨秀伟.生物碱[M].北京:化学工业出版社,2005.Yang XW. Alkaloid[M]. Beijing:Chemical Industry Press, 2005.
[11]张轩,周斌,路慧丽.草乌甲素的药理作用机制及临床应用研究进展[J].慢性病学杂志,2016,(11):1210-1213.Zhang X, Zhou B, Lu HL. Advances in pharmacological mechanism and clinical application of aconitine[J]. Chron Pathematol J, 2016,(11):1210-1213.
[12]邓鸣,蒋受军,朱斌.飞行时间质谱分析技术在植物药研究中的应用进展[J].华西药学杂志,2013, 28(1):12-15.Deng M, Jiang SJ, Zhu B. Progress in the application of time-of-flight mass spectrometry in the study of botanical drugs[J]. West Chin J Pharm Sci, 2013, 28(1):12-15.
[13] Jiang T, Jiang Z, Mukadaisi S, et al. Research progress in hydrolysis mechanism and LC-MS determination method of genus Aconitum L. based on traditional Chinese medicine simulation processing[J]. Chin Trad Herb Drug, 2016,(12):121-130.
[14] GA/T934-2011生物样品中次乌头碱、乌头碱、中乌头碱的液相色谱-串联质谱检验方法[S].GA/T 934-2011 Determination of sub aconitine, aconitine and meconoxine in biological samples by liquid chromatography-tandem mass spectrometry[S].
[15] Lei S, Wang SC, Sun MQ, et al. Fragmentation pathways of six aconitine alkaloids using HPLC-Q-TOF-MS technology[J]. Chin Trad Herb Drug,2016,(16):2827-2828.
[16]欧水平.基于“一体多用”的乌头新药用部位及制剂研究-川乌头茎叶治疗类风湿性关节炎的成药性研究[D].成都:成都中医药大学,2013.Ou SP. New part and preparation for medicine of aconitum carmichaeli debx. based on"A integrated plant but multi-medicine"-the druggability research of leaf and stem in treating rheumatoid arthritis[D]. Chengdu:Chengdu University of Traditional Chinese Medicine, 2013.
[17]蒋荡,侯大斌,邓文伟,等.不同产地附子中附子灵和宋果灵含量的比较[J].北方园艺,2013,(6):159-161.Jiang D, Hou DB, Deng WW, et al. Study on content comparison of fuziline and songorine from daughter roots of aconitum carmicichaeli produced from different areas[J]. Northern Horticult, 2013,(6):159-161.
[18]杨杏林,杨大男,杨清锐,等.草乌甲素的研究进展[J].世界临床药物,2013,34(2):80-83.Yang XL, Yang DN, Yang QR, et al. Research advances of bulleyaconitine A[J]. World Clin Drug, 2013, 34(2):80-83.