超高效二维液相色谱法测定复方南星止痛膏中双酯型乌头生物碱的含量
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摘要
目的:采用在线中心切割超高效二维液相色谱(2D-UPLC)法,建立同时测定复方南星止痛膏中3个双酯型乌头生物碱(新乌头碱、乌头碱和次乌头碱)的含量测定方法。方法:一维UPLC采用Waters XBridgeBEH C_(18)色谱柱(2.1 mm×50 mm 1.7μm),以甲醇-0.008%二乙胺为流动相,梯度洗脱,流速0.1 mL·min~(-1),柱温18℃;二维UPLC采用Waters XBridge BEH C_(18)色谱柱(3.0 mm×100 mm 1.7μm),以乙腈-0.01%磷酸为流动相,梯度洗脱,流速0.7 mL·min~(-1),柱温40℃。进样量1μL,检测波长235 nm。结果:新乌头碱、乌头碱和次乌头碱的线性范围分别为0.66～9.90、0.55～8.20和0.91～13.72μg·mL~(-1),R2分别为0.999 5、0.999 9和0.999 6,线性关系良好;3个成分的平均加样回收率在82.5%～88.8%之间,方法的准确度和稳定性均较好;复方南星止痛膏中新乌头碱、乌头碱和次乌头碱含量分别在3.2～5.0、0.9～1.3、8.5～10.4μg·g~(-1)范围内。结论:该方法有效改善了复方南星止痛膏中待测组分的分离度,极大提高了检测灵敏度,对制剂中的3个双酯型乌头生物碱进行准确定量分析,提高产品的质量控制水平,并为临床用药提供理论支持。
Objective:To establish an on-line center-cutting two-dimensional ultra performance liquid chromategraphy(2 D-UPLC)method for the determination of three double ester alkaloids(mesaconitine,aconitine and hypaconitine)in compound Nanxing Zhitong plaster.Methods:The first dimensional column was Waters XBridge BEH C_(18)(2.1 mm×50 mm 1.7 μm),with methanol and 0.008% diethylamine as the mobile phase in gradient elution at a flow rate of 0.1 mL·min~(-1),and the column temperature was 18 ℃.The second dimensional column was Waters XBridge BEH C_(18)(3.0 mm×100 mm 1.7 μm),with acetonitrile-0.01% phosphoric acid as the mobile phase in gradient elution at a flow rate of 0.7 mL·min~(-1),and the column temperature was 40 ℃.The injection volume was 1 μL and the detection wavelength was 235 nm.Results:The linear ranges of mesaconitine,aconitine and hypaconitine were 0.66-9.90μg·mL~(-1),0.55-8.20 μg·mL~(-1) and 0.91-13.72 μg·mL~(-1) with R~2 of 0.999 5,0.999 9 and 0.999 6,respectively,which indicated good linear relationship.The average recoveries of the three compounds were 82.5%-88.8%.The contents of mesaconitine, aconitine and hypaconitine in the compound Nanxing Zhitong plaster were within the range of 3.2-5.0μg·g~(-1), 0.9-1.3 μg·g~(-1) and 8.5-10.4 μg·g~(-1).Conclusion:It was proved that the method could effectively improve separation and sensitivity,and could determine the three diester alkaloids in an accurate way.The investigation provided theoretical basis for enhancement of quality control of compound Nanxing Zhitong plaster and for clinic application.
Objective:To establish an on-line center-cutting two-dimensional ultra performance liquid chromategraphy(2 D-UPLC)method for the determination of three double ester alkaloids(mesaconitine,aconitine and hypaconitine)in compound Nanxing Zhitong plaster.Methods:The first dimensional column was Waters XBridge BEH C_(18)(2.1 mm×50 mm 1.7 μm),with methanol and 0.008% diethylamine as the mobile phase in gradient elution at a flow rate of 0.1 mL·min~(-1),and the column temperature was 18 ℃.The second dimensional column was Waters XBridge BEH C_(18)(3.0 mm×100 mm 1.7 μm),with acetonitrile-0.01% phosphoric acid as the mobile phase in gradient elution at a flow rate of 0.7 mL·min~(-1),and the column temperature was 40 ℃.The injection volume was 1 μL and the detection wavelength was 235 nm.Results:The linear ranges of mesaconitine,aconitine and hypaconitine were 0.66-9.90μg·mL~(-1),0.55-8.20 μg·mL~(-1) and 0.91-13.72 μg·mL~(-1) with R~2 of 0.999 5,0.999 9 and 0.999 6,respectively,which indicated good linear relationship.The average recoveries of the three compounds were 82.5%-88.8%.The contents of mesaconitine, aconitine and hypaconitine in the compound Nanxing Zhitong plaster were within the range of 3.2-5.0μg·g~(-1), 0.9-1.3 μg·g~(-1) and 8.5-10.4 μg·g~(-1).Conclusion:It was proved that the method could effectively improve separation and sensitivity,and could determine the three diester alkaloids in an accurate way.The investigation provided theoretical basis for enhancement of quality control of compound Nanxing Zhitong plaster and for clinic application.
引文
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[6]罗春梅,易凡力,黄志芳,等.HPLC-ELSD法测定附子水溶性生物碱提取物和蒸附片中5种生物碱的含量[J].药物分析杂志,2018,38(6):935LUO CM,YI FL,HUANG ZF,et al.Determination of five representative components in the extract of water-soluble alkaloids from Aconiti Lateralis Radix Praeparata and Zhengfu tablets by HPLC-ELSD method[J].Chin J Pharm Anal,2018(6):935
[7]黄志芳,易进海,陈东安,等.制川乌HPLC特征图谱研究和6种酯型生物碱的含量测定[J].药物分析杂志,2011,31(2):217HUANG ZF,YI JH,CHEN DA,et al.HPLC specific chromatogram of Radix Aconiti Praeparata and determination of six esoteric alkaloids[J].Chin J Pharm Anal,2011,31(2):217
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[9]张艳海,金燕,王峥涛.在线二维多中心切割液相色谱法测定三七、人参及其相关产品中8种人参皂苷[J].中草药,2017,48(5):894ZHANG YH,JIN Y,WANG ZT.Simultaneous quantitation of eight ginsenosides in Notoginseng Radix et Rhizoma,Ginseng Radix et Rhizoma,and their related products by online multi heart-cutting two dimensional liquid chromatography[J].Chin Tradit Herb Drugs,2017,48(5):894
[10]张艳海,张大伟,孟兆青,等.在线二维液相色谱法快速测定桂枝茯苓胶囊中芍药苷、丹皮酚、苦杏仁苷和肉桂酸的含量[J].中国中药杂志,2013,38(23):4088ZHANG YH,ZHANG DW,MENG ZQ,et al.Rapid determination of four components in Guizhi Fuling capsule with online two-dimensional liquid chromatography[J].China J Chin Mater Med,2013,38(23):4088
[11]赵雅清,付冬梅,刘艳芳,等.离线二维液相色谱法分离巴天酸模根的化学成分[J].色谱,2018,36(1):37ZHAOYQ,FUDM,LIUYF,etal.Separationofchemical compositions in root of Rumex patientia L.with off-line twodimensional liquid chromatography[J].Chin J Chromatogr,2018,36(1):37
[12]国金玲,连云岚,张远芳,等.中心切割-二维液相色谱分离清热解毒片(胶囊)中的咖啡酰奎宁酸类成分[J].中国药品标准,2017,18(6):449GUO JL,LIAN YL,ZHANG YF,et al.Separation of caffeoylquinic acids in Qingrejiedu tablets(capsules)by central-cutting twodimensional HPLC[J].Drug Stand China,2017,18(6):449
[13]廖强,罗达龙,韦日伟,等.在线二维液相色谱法同时测定银翘解毒丸中5种成分[J].中药材,2017,40(12):2891LIAO Q,LUO DL,WEI RW,et al.On-line two-dimensional liquid chromatography for the simultaneous determination of 5 kinds of components in Yinqiaojiedu pills[J].J Chin Med Mater,2017,40(12):2891
[14]黄竞怡,佟玲,丁黎.二维液相色谱在中药分析的应用[J].药学进展,2015,39(5):357HUANG JY,TONG L,DING L.Application of two-dimensional liquid chromatography in analysis of traditional Chinese medicines[J].Prog Pharm Sci,2015,39(5):357
[15]杨倩,王志英,唐素芳.二维超高效液相色谱-QTof质谱联用技术在盐酸博来霉素杂质谱研究中的应用[J].药物分析杂志,2016,36(7):1231YANG Q,WANG ZY,TANG SF.Application of two-dimensional UPLC-QTof MS technology in the study of the impurity profile of bleomycin hydrochloride[J].Chin J Pharm Anal,2016,36(7):1231
[16]高辉,温学森,马小军,等.二维液相色谱技术在药物分析中的应用[J].药物分析杂志,2007,27(4):616GAO H,WEN XS,MA XJ,et al.The technique of two-dimensinoal liquid chromatography(2D-LC)and its applications in drug analysis[J].Chin J Pharm Anal,2007,27(4):616
[17]王瑞,刘芳,孙毅坤,等.不同附子炮制品中乌头碱、新乌头碱、次乌头碱含量的HPLC测定[J].药物分析杂志,2006,26(10):1361WANG R,LIU F,SUN YK,et al.Quantitative analysis of aconitine,mesaconitine,hypaconitine in different Radix Aconiti Lateralis Praeparata[J].Chin J Pharm Anal,2006,26(10):1361
[18]郭爱华.川乌、附子的毒性和药理作用[J].山西职工医学院学报,1995,5(2):65GUO AH.Toxicology and pharmacological effects of Aconitum alksloids and Aconiti Lateralis Radix Praeparata[J].J Shanxi Med Coll,1995,5(2):65
[19]王蕾,张先灵,王鑫,等.UPLC-MSn法对生川乌、生附片化学成分的比较研究[J].药物分析杂志,2017,37(9):1640WANG L,ZHANG XL,WANG X,et al.Comparative study on unprocessed Anconiti Radix and unprocessed Aconiti Lateralis Radix by UPLC-MS[J].Chin J Pharm Anal,2017,37(9):1640
[20]张聿梅,鲁静,蒋渝,等.川乌和制川乌中单酯及双酯型生物碱成分的含量测定[J].药物分析杂志,2005,25(7):807ZHANG YM,LU J,JIANG Y,et al.HPLC determination of Aconitum alkaloids and their hydrolysis products in Radix Aconiti and its preparate[J].Chin J Pharm Anal,2005,25(7):807
[2]胡晨,陈荣明,殷书梅,等.复方南星止痛膏的镇痛作用观察及机理探讨[J].南京中医药大学学报,2009,25(2):140HU C,CHEN RM,YIN SM,et al.The approaches onanalgesic effect and mechanisms of Nanxing Zhitong plaster[J].J Nanjing Univ TCM,2009,25(2):140
[3]刘为民,陆茵,许慧琪.复方南星止痛膏止痛机理研究[J].江苏中医,1998,19(10):46LIU WM,LU Y,XU HQ.Study on mechanism of pain of compound Nanxing Zhitong plaster[J].Jiangsu J Tradit Chin Med,1998,19(10):46
[4]卞慧敏,俞晶华,姜淼,等.复方南星止痛膏抗炎作用研究[J].中药药理与临床,2007,23(5):164BIAN HM,YU JH,JIANG M,et al. Studies onanti-inflammatory of compound Nanxing Zhitong plaster[J].Pharmacol Clin Chin Mater Clin Med,2007,23(5):46
[5]中华人民共和国药典2015年版.一部[S].2015:39ChP 2015.VolⅠ[S].2015:39
[6]罗春梅,易凡力,黄志芳,等.HPLC-ELSD法测定附子水溶性生物碱提取物和蒸附片中5种生物碱的含量[J].药物分析杂志,2018,38(6):935LUO CM,YI FL,HUANG ZF,et al.Determination of five representative components in the extract of water-soluble alkaloids from Aconiti Lateralis Radix Praeparata and Zhengfu tablets by HPLC-ELSD method[J].Chin J Pharm Anal,2018(6):935
[7]黄志芳,易进海,陈东安,等.制川乌HPLC特征图谱研究和6种酯型生物碱的含量测定[J].药物分析杂志,2011,31(2):217HUANG ZF,YI JH,CHEN DA,et al.HPLC specific chromatogram of Radix Aconiti Praeparata and determination of six esoteric alkaloids[J].Chin J Pharm Anal,2011,31(2):217
[8]沈保家,秦昆明,刘启迪,等.二维色谱技术及其在中药领域中的应用[J].中国科学:化学,2013,43(11):1480SHEN BJ,QIN KM,LIU QD,et al.Two-dimensional chromatography technology and its applications in traditional Chinese medicine[J].Sci Sin Chim,2013,43(11):1480
[9]张艳海,金燕,王峥涛.在线二维多中心切割液相色谱法测定三七、人参及其相关产品中8种人参皂苷[J].中草药,2017,48(5):894ZHANG YH,JIN Y,WANG ZT.Simultaneous quantitation of eight ginsenosides in Notoginseng Radix et Rhizoma,Ginseng Radix et Rhizoma,and their related products by online multi heart-cutting two dimensional liquid chromatography[J].Chin Tradit Herb Drugs,2017,48(5):894
[10]张艳海,张大伟,孟兆青,等.在线二维液相色谱法快速测定桂枝茯苓胶囊中芍药苷、丹皮酚、苦杏仁苷和肉桂酸的含量[J].中国中药杂志,2013,38(23):4088ZHANG YH,ZHANG DW,MENG ZQ,et al.Rapid determination of four components in Guizhi Fuling capsule with online two-dimensional liquid chromatography[J].China J Chin Mater Med,2013,38(23):4088
[11]赵雅清,付冬梅,刘艳芳,等.离线二维液相色谱法分离巴天酸模根的化学成分[J].色谱,2018,36(1):37ZHAOYQ,FUDM,LIUYF,etal.Separationofchemical compositions in root of Rumex patientia L.with off-line twodimensional liquid chromatography[J].Chin J Chromatogr,2018,36(1):37
[12]国金玲,连云岚,张远芳,等.中心切割-二维液相色谱分离清热解毒片(胶囊)中的咖啡酰奎宁酸类成分[J].中国药品标准,2017,18(6):449GUO JL,LIAN YL,ZHANG YF,et al.Separation of caffeoylquinic acids in Qingrejiedu tablets(capsules)by central-cutting twodimensional HPLC[J].Drug Stand China,2017,18(6):449
[13]廖强,罗达龙,韦日伟,等.在线二维液相色谱法同时测定银翘解毒丸中5种成分[J].中药材,2017,40(12):2891LIAO Q,LUO DL,WEI RW,et al.On-line two-dimensional liquid chromatography for the simultaneous determination of 5 kinds of components in Yinqiaojiedu pills[J].J Chin Med Mater,2017,40(12):2891
[14]黄竞怡,佟玲,丁黎.二维液相色谱在中药分析的应用[J].药学进展,2015,39(5):357HUANG JY,TONG L,DING L.Application of two-dimensional liquid chromatography in analysis of traditional Chinese medicines[J].Prog Pharm Sci,2015,39(5):357
[15]杨倩,王志英,唐素芳.二维超高效液相色谱-QTof质谱联用技术在盐酸博来霉素杂质谱研究中的应用[J].药物分析杂志,2016,36(7):1231YANG Q,WANG ZY,TANG SF.Application of two-dimensional UPLC-QTof MS technology in the study of the impurity profile of bleomycin hydrochloride[J].Chin J Pharm Anal,2016,36(7):1231
[16]高辉,温学森,马小军,等.二维液相色谱技术在药物分析中的应用[J].药物分析杂志,2007,27(4):616GAO H,WEN XS,MA XJ,et al.The technique of two-dimensinoal liquid chromatography(2D-LC)and its applications in drug analysis[J].Chin J Pharm Anal,2007,27(4):616
[17]王瑞,刘芳,孙毅坤,等.不同附子炮制品中乌头碱、新乌头碱、次乌头碱含量的HPLC测定[J].药物分析杂志,2006,26(10):1361WANG R,LIU F,SUN YK,et al.Quantitative analysis of aconitine,mesaconitine,hypaconitine in different Radix Aconiti Lateralis Praeparata[J].Chin J Pharm Anal,2006,26(10):1361
[18]郭爱华.川乌、附子的毒性和药理作用[J].山西职工医学院学报,1995,5(2):65GUO AH.Toxicology and pharmacological effects of Aconitum alksloids and Aconiti Lateralis Radix Praeparata[J].J Shanxi Med Coll,1995,5(2):65
[19]王蕾,张先灵,王鑫,等.UPLC-MSn法对生川乌、生附片化学成分的比较研究[J].药物分析杂志,2017,37(9):1640WANG L,ZHANG XL,WANG X,et al.Comparative study on unprocessed Anconiti Radix and unprocessed Aconiti Lateralis Radix by UPLC-MS[J].Chin J Pharm Anal,2017,37(9):1640
[20]张聿梅,鲁静,蒋渝,等.川乌和制川乌中单酯及双酯型生物碱成分的含量测定[J].药物分析杂志,2005,25(7):807ZHANG YM,LU J,JIANG Y,et al.HPLC determination of Aconitum alkaloids and their hydrolysis products in Radix Aconiti and its preparate[J].Chin J Pharm Anal,2005,25(7):807