辽细辛中十二碳四烯酰胺A，B的定性及定量分析研究(1)
[摘要]建立辽细辛中2个主要的直链酰胺成分十二碳四烯酰胺A与十二碳四烯酰胺B的定性与定量分析方法，并测定其在42份辽细辛(37份不同年份收集的北细辛及5份汉城细辛)中的含量。利用HPLC-IT-TOF-MS/MS技术结合对照品鉴定北细辛甲醇提取液中的十二碳四烯酰胺A与B;对2种成分的含量测定采用超高效液相色谱-二极管阵列检测器(UPLC-PDA)，ACQUITY UPLC BEH C18色谱柱(2.1 mm×100 mm，1.7 μm)，流动相为纯水-乙腈，梯度洗脱，柱温45 ℃，检测波长254 nm。结果表明，HPLC-IT-TOF-MS/MS鉴别细辛中十二碳四烯酰胺A、B的准分子离子[M+H]+为m/z 248.20;2种成分在UPLC-PDA上分离度良好，在检测范围内呈良好线性，平均加样回收率为97.90%和99.86%。在所测定辽细辛样品中，十二碳四烯酰胺A的含量为0.11～3.89 mg·g-1，十二碳四烯酰胺B为0.24～6.65 mg·g-1。含量测定结果显示随着贮藏时间的延长，十二碳四烯酰胺A与十二碳四烯酰胺B的含量均呈现降低的趋势，与2013年收集样品相比，2002～2003年收集样品中两者的平均含量分别降低34%和36%;这2种成分在汉城细辛中的平均含量[十二碳四烯酰胺A：(0.78±0.52)mg·g-1;十二碳四烯酰胺B：(1.69±0.83) mg·g-1]均显著低于北细辛[十二碳四烯酰胺A：(1.59±0.75)mg·g-1;十二碳四烯酰胺B：(2.90±1.17)mg·g-1](P<0.05);辽细辛地上部分十二碳四烯酰胺A的含量为0.11～0.33 mg·g-1，十二碳四烯酰胺B含量为0.24～0.60 mg·g-1，两者的含量均明显低于同一植株的地下部分(分别为0.73～3.89，2.11 ～6.24 mg·g-1)。本方法快速简便、结果准确，对2种直链酰胺类成分可达到良好的分离并满足含量测定的要求，可用于辽细辛药材中这2种成分的定性与定量分析，为辽细辛药材质量控制方法的进一步提高提供依据。
, 百拇医药
[关键词]辽细辛;HPLC-IT-TOF-MS/MS;UPLC;十二碳四烯酰胺A;十二碳四烯酰胺B
Qualitative and quantitative analysis of dodecatetraenamides
A，B in Asari Radix et Rhizoma
XIE De-mei 1， LIU Guang-xue 1， XU Feng 1， SHANG Ming-ying 1， ZHANG Zi-wei 2， WANG Xuan 2， CAI Shao-qing 1*
(1. State Key Laboratory of Natural and Biomimetic Drugs， School of Pharmaceutical Sciences， Peking University，Beijing 100191， China;
, 百拇医药
2.Department of Chemical Biology， School of Pharmaceutical Sciences， Peking University，Beijing 100191， China)
[Abstract]To develop an analytic method for qualitative and quantitative analysis of dodecatetraenamides A，B in 42 samples of two official species of Asari Radix et Rhizoma(ARR)(37 samples of Asarum heterotropoides var. mandshuricum with different collection time and 5 samples of Asarum sieboldiivar.seoulense). The HPLC-IT-TOF-MS/MS methods for the qualitative and UPLC-PDA methods for the quantitative analysis were established.Dodecatetraenamides A，B were identified by comparing the retention time，UV absorption spectrum and quasi-molecular ion peak [M+H]+ with the reference compound using HPLC-IT-TOF-MS/MS. The content of dodecatetraenamides A and B in ARR were determined by UPLC-PDA. The separation was successfully carried out on a ACQUITY UPLC BEH C18(2.1 mm×100 mm，1.7 μm)column eluted with mobile phases of water(A) and acetonitrile(B) in gradient program( 0-3 min，35%B; 3-5 min，35%-36%B; 5-6 min，36%-43%B; 6 min-11 min 43%B;11-12 min，43%-100%B). The column temperature was 45 °C，and the detection wavelength was set at 254 nm. The flow rate was 0.6 mL· min-1.On one level mass spectrometry scanning， the results showed that the quasi-molecular ion [M+H]+ of both dodecatetraenamides A and B were m/z 248.20. The quantitative method with UPLC-PDA has made the baseline separation of the constituents， which were reported as mixtures in the most literatures.The average recovery of dodecatetraenamides A and B were 97.90% and 99.86%，the relative standard deviation were 0.4% and 1.1%， respectively.The contents of dodecatetraenamides A，B in all ARR samples was in the range of 0.11-3.89 and 0.24-6.65 mg·g-1.Their contents reduced with the extension of storage time. Compared with the samples of 2013， the average content of the two constituents in the samples collected in year 2002-2003 reduced 34% and 36%，respectively(P<0.05).Compared the A. sieboldii var. seoulense and A. heterotropoides var. mandshuricum with the same collective time and production area， the average contents of the two constituents in latter were up to(1.59±0.75) mg·g-1 and(2.90±1.17) mg·g-1， respectively， significantly higher than that in A. sieboldii var. seoulense (dodecatetraenamide A were(0.78±0.52) mg·g-1， dodecatetraenamide B were(1.69±0.83) mg·g-1)(P<0.05). The content of the dodecatetraenamide A in overground part was in the range of 0.11-0.33 mg·g-1， dodecatetraenamide B was 0.24-0.60 mg·g-1 ， which were much lower than that of the underground part of ARR(dodecatetraenamide A was in the range of 0.73-3.89 mg·g-1， dodecatetraenamide B was 2.11-6.24 mg·g-1). The method was certified to be simple， accurate and reliable and could be used for qualitative and quantitative analysis of dodecatetraenamide A and B in different species of ARR， also can be used for the comprehensive quality control of traditional Chinese medicine， Asari Radix et Rhizoma., 百拇医药(谢德媚 刘广学 徐风 尚明英 张子为 王璇 蔡少青)
, 百拇医药
[关键词]辽细辛;HPLC-IT-TOF-MS/MS;UPLC;十二碳四烯酰胺A;十二碳四烯酰胺B
Qualitative and quantitative analysis of dodecatetraenamides
A，B in Asari Radix et Rhizoma
XIE De-mei 1， LIU Guang-xue 1， XU Feng 1， SHANG Ming-ying 1， ZHANG Zi-wei 2， WANG Xuan 2， CAI Shao-qing 1*
(1. State Key Laboratory of Natural and Biomimetic Drugs， School of Pharmaceutical Sciences， Peking University，Beijing 100191， China;
, 百拇医药
2.Department of Chemical Biology， School of Pharmaceutical Sciences， Peking University，Beijing 100191， China)
[Abstract]To develop an analytic method for qualitative and quantitative analysis of dodecatetraenamides A，B in 42 samples of two official species of Asari Radix et Rhizoma(ARR)(37 samples of Asarum heterotropoides var. mandshuricum with different collection time and 5 samples of Asarum sieboldiivar.seoulense). The HPLC-IT-TOF-MS/MS methods for the qualitative and UPLC-PDA methods for the quantitative analysis were established.Dodecatetraenamides A，B were identified by comparing the retention time，UV absorption spectrum and quasi-molecular ion peak [M+H]+ with the reference compound using HPLC-IT-TOF-MS/MS. The content of dodecatetraenamides A and B in ARR were determined by UPLC-PDA. The separation was successfully carried out on a ACQUITY UPLC BEH C18(2.1 mm×100 mm，1.7 μm)column eluted with mobile phases of water(A) and acetonitrile(B) in gradient program( 0-3 min，35%B; 3-5 min，35%-36%B; 5-6 min，36%-43%B; 6 min-11 min 43%B;11-12 min，43%-100%B). The column temperature was 45 °C，and the detection wavelength was set at 254 nm. The flow rate was 0.6 mL· min-1.On one level mass spectrometry scanning， the results showed that the quasi-molecular ion [M+H]+ of both dodecatetraenamides A and B were m/z 248.20. The quantitative method with UPLC-PDA has made the baseline separation of the constituents， which were reported as mixtures in the most literatures.The average recovery of dodecatetraenamides A and B were 97.90% and 99.86%，the relative standard deviation were 0.4% and 1.1%， respectively.The contents of dodecatetraenamides A，B in all ARR samples was in the range of 0.11-3.89 and 0.24-6.65 mg·g-1.Their contents reduced with the extension of storage time. Compared with the samples of 2013， the average content of the two constituents in the samples collected in year 2002-2003 reduced 34% and 36%，respectively(P<0.05).Compared the A. sieboldii var. seoulense and A. heterotropoides var. mandshuricum with the same collective time and production area， the average contents of the two constituents in latter were up to(1.59±0.75) mg·g-1 and(2.90±1.17) mg·g-1， respectively， significantly higher than that in A. sieboldii var. seoulense (dodecatetraenamide A were(0.78±0.52) mg·g-1， dodecatetraenamide B were(1.69±0.83) mg·g-1)(P<0.05). The content of the dodecatetraenamide A in overground part was in the range of 0.11-0.33 mg·g-1， dodecatetraenamide B was 0.24-0.60 mg·g-1 ， which were much lower than that of the underground part of ARR(dodecatetraenamide A was in the range of 0.73-3.89 mg·g-1， dodecatetraenamide B was 2.11-6.24 mg·g-1). The method was certified to be simple， accurate and reliable and could be used for qualitative and quantitative analysis of dodecatetraenamide A and B in different species of ARR， also can be used for the comprehensive quality control of traditional Chinese medicine， Asari Radix et Rhizoma., 百拇医药(谢德媚 刘广学 徐风 尚明英 张子为 王璇 蔡少青)