基于液质联用技术的大小蓟多组分分析与黄酮类成分的药物代谢动力学研究
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摘要
大蓟为菊科蓟属植物蓟Cirsium joponicum DC.的干燥地上部分，小蓟为菊科蓟属植物刺儿菜Cirsium setosum（Willd.）MB.的干燥地上部分，两种药材在中国大部分地区均有分布。具有凉血止血，祛瘀消肿的功能，是《中国药典》2010版收载的常用中药。现代药理研究发现大小蓟具有止血、抗菌、抗肿瘤和抗微生物等作用。大小蓟化学成分复杂，现有研究报道的化学成分主要可分为黄酮及黄酮苷类、酚酸、甾醇、长链烯炔醇、生物碱及一些其他类化合物。目前的化学成分研究和药理学研究揭示了黄酮和酚酸类成分为其止血、抗炎的主要活性成分。
迄今为止国内外学者对大小蓟的研究主要集中在化学成分鉴定与分离,而对于其质量评价与体内药代动力学研究涉及较少。本文采用LC-ESI-MS/MS技术对大小蓟中的黄酮和酚酸类成分同时进行定性及定量研究，为中药材大蓟和小蓟的质量控制提供新的分析手段。且研究了小蓟中7种活性成分在动物体内的动力学过程和排泄规律，对中药材小蓟的临床应用提供依据。
第一部分液质联用法同时测定小蓟中11种活性成分的含量及化学计量学分析
目的：建立一种快速、准确的LC-ESI-MS/MS定量分析方法，可同时分离、测定小蓟中11种活性成分（10种黄酮，包括蒙花苷、刺槐素、芦丁、香叶木素、高车前素、芹菜素、柚皮素、橙皮苷、木犀草素和槲皮素；1种酚酸，原儿茶酸），并用于小蓟药材的质量分析。采用主成分分析及聚类分析的统计方法分类和区分25批不同产地的小蓟药材。
方法：液相色谱柱为Diamonsil C_(18)柱（150×4.6mm,5μm），流动相为甲醇：水（含有0.1‰乙酸），流速为0.8mL/min，分析时间为19min，梯度洗脱。质谱采用电喷雾离子源（ESI源），TurboV离子源，采用多反应离子监测（MRM），进行正负离子同时检测。源喷射电压（IS）为5500V和-4500V，雾化温度为650℃，雾化气（GS1，N_2）为60psi，辅助气（GS2，N_2）为65psi，气帘气（N_2）为25psi。实验采用SPSS统计软件对数据进行统计分析。
结果：11种活性成分的线性关系良好（r~2≥0.9941），检测限和定量限分别小于3.96ng/mL和9.90ng/mL，日内日间精密度分别小于3.30%和3.57%。平均回收率范围在96.4%～104.2%，48h内的稳定性良好。主成分分析及聚类分析对25批小蓟药材成功分类区分，证明河北产药材质量最佳。
结论：本法简便、快速、灵敏度高、专属性好，可用于小蓟药材中蒙花苷、刺槐素、芦丁、香叶木素、高车前素、芹菜素、柚皮素、橙皮苷、木犀草素、槲皮素和原儿茶酸的含量测定，为小蓟药材的质量控制提供了新的方法和手段。
第二部分液质联用法同时测定大鼠口服小蓟提取物后血浆中7种成分及药代动力学研究
目的：建立一种同时测定大鼠血浆中蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸含量的LC-ESI-MS/MS方法，并成功的用于大鼠灌胃给予小蓟提取物后血浆中7种成分的药代动力学研究，并建立其药-时曲线，阐明药动学参数与特征。
方法：大鼠灌胃给与小蓟提取物（8mL/kg）后，分别于给药后0.17，0.5，1，1.5，2，3，4，6，8，12，24和36h眼内眦静脉丛取血0.3mL，制备血浆样品，采用乙酸乙酯液-液提取方法进行样品预处理，磺胺甲噁唑为内标。反相Diamonsil C_(18)柱（150×4.6mm,5μm），流动相为A（甲醇）-B（0.1‰乙酸水），梯度洗脱：0.0-1.5min，35%-60%A；1.5-10.0min，60%-63%A；10.0-10.1min，63%-95%A；10.1-15.0min，95%A等度洗脱。进样前预平衡6min，柱温为30℃，流速为0.8mL/min。质谱条件：ESI源，源电压（IS）5500V和-4500V，源温度（TEM）650℃，雾化气（GS1，N_2）60psi，辅助气（GS2，N_2）65psi，气帘气（N_2）25psi。采用电喷雾离子源（ESI），多周期正负离子同时扫描，多反应监测（MRM）模式进行定量。7种被测成分和内标物的监测离子对分别为蒙花苷m/z593.3/285.3、刺槐素m/z283.1/267.9、芦丁m/z609.1/299.9、橙皮苷m/z609.3/300.9、木犀草素m/z284.9/133.1、芹菜素m/z269.0/117.0、原儿茶酸m/z153.0/108.9和磺胺甲噁唑m/z252.0/156.0。
结果：血浆中蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸分别在1.87～935、2.63～1315、7.80～3900、2.68～1340、1.99～995、1.53～765和2.84～1420ng/mL范围内线性关系良好（r~2≥0.9954），最低定量限（LLOQ）≤7.80ng/mL。日内、日间精密度的相对标准偏差（RSD）均小于9.4%，相对误差（RE）为-4.8%～9.8%。平均提取回收率为71.0%～89.7%。大鼠口服灌胃给予小蓟提取物后，血浆中7种待测成分蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸在大鼠体内的药代动力学药-时曲线的有所差异。其中，蒙花苷和原儿茶酸吸收最快，达峰时间为给药后2h左右；芦丁、橙皮苷、木犀草素和芹菜素吸收稍慢，达峰时间为给药后3h左右；刺槐素吸收最慢，于给药后6h达到血浆峰浓度。总体来说，大鼠灌胃给予小蓟提取物后，7种待测成分在体内吸收均较迅速，10min均可检测到，并且具有相近的消除速率。
结论：本研究建立了LC-ESI-MS/MS法同时测定大鼠血浆中蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸的含量，可用于小蓟中7种活性成分在大鼠体内的药代动力学研究。该方法简便、快速、灵敏度高、专属性好，对小蓟药材的体内定量分析具有重要意义，有益于传统中药小蓟的临床应用。
第三部分液质联用法同时测定大鼠口服小蓟提取物后胆汁和尿液中7种成分及排泄动力学研究
目的：建立LC-ESI-MS/MS同时测定大鼠胆汁和尿液中7种成分（蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸）含量的方法，并用于大鼠灌胃给予小蓟提取物后该7种成分的胆汁和尿液排泄动力学研究。
方法：大鼠灌胃给予小蓟提取物8mL/kg，胆汁按照0-2，2-4，4-6，6-8，8-12，12-24，24-30，30-36h收集，尿液按照0-4，4-8，8-12，12-24，24-36，36-48，48-60，60-72，72-84h时间段收集。采用乙酸乙酯液-液提取方法进行样品前处理，磺胺甲噁唑为内标。反相Diamonsil C_(18)柱（150×4.6mm,5μm），流动相为A（甲醇）-B（0.1‰乙酸水），梯度洗脱：0.0-1.5min，35%-60%A；1.5-10.0min，60%-63%A；10.0-10.1min，63%-95%A；10.1-15.0min，95%A等度洗脱。进样前预平衡6min，柱温为30℃，流速为0.8mL/min。质谱条件：ESI源，源电压（IS）5500V和-4500V，源温度（TEM）650℃，雾化气（GS1，N_2）60psi，辅助气（GS2，N_2）65psi，气帘气（N_2）25psi。采用电喷雾离子源（ESI），多周期正负离子同时扫描，多反应监测（MRM）模式进行定量。7种被测成分和内标物的监测离子对分别为蒙花苷m/z593.3/285.3、刺槐素m/z283.1/267.9、芦丁m/z609.1/299.9、橙皮苷m/z609.3/300.9、木犀草素m/z284.9/133.1、芹菜素m/z269.0/117.0、原儿茶酸m/z153.0/108.9和磺胺甲噁唑m/z252.0/156.0。测定大鼠灌胃给予小蓟提取物后胆汁和尿液中7种成分的累积排泄率。
结果：胆汁和尿液中7种活性成分在测定浓度范围内均具有良好的线性关系（r~2≥0.9930），日内日间精密度均小于9.6%，相对误差RE均在±15%内。在胆汁样品中提取回收率为65.0%～81.2%，基质效应为85.4%～104.6%。尿液样品中提取回收率为71.0%～83.6%，基质效应为92.0%～104.7%。胆汁及尿液样品稳定性良好。试验结果表明，大鼠的胆汁中检测到蒙花苷、刺槐素、芦丁、橙皮苷、芹菜素和原儿茶酸6个分析物，它们的累计排泄率分别为0.499，0.088，0.163，0.283，0.146和0.338%，6个分析物36h基本排泄完全；大鼠的尿液中检测到蒙花苷、刺槐素、橙皮苷、木犀草素和芹菜素5个分析物，其累积排泄率分别为0.015，1.164，0.329，0.201和2.182%，5个分析物84h基本排泄完全。。
结论：本研究首次采用LC-ESI-MS/MS法测定了大鼠胆汁和尿液中小蓟中7种活性成分，并研究了其排泄动力学。方法准确，结果可靠，可满足中药材在生物基质中的排泄研究。该法对小蓟药材的进一步临床应用具有重要意义。
第四部分液质联用技术同时测定大蓟中13种成分及黄酮类成分的裂解规律分析
目的：建立一种快速、准确的LC-ESI-MS/MS定量分析方法，可同时分离、测定大蓟中13种成分（12种黄酮，包括柳穿鱼叶苷、柳穿鱼黄素、蒙花苷、刺槐素、芦丁、香叶木素、高车前素、芹菜素、柚皮素、橙皮苷、木犀草素和槲皮素；1种酚酸，原儿茶酸），并用于大蓟药材分析。采用ESI-MS/MS法推断大蓟药材中12种黄酮类化合物的裂解规律。
方法：液相色谱柱为Diamonsil C_(18)柱（150×4.6mm,5μm），流动相为甲醇：水（含有0.1‰乙酸），流速为0.8mL/min，分析时间为19min，梯度洗脱。质谱采用电喷雾离子源（ESI源），TurboV离子源，采用多反应离子监测（MRM），进行正负离子同时检测。源喷射电压（IS）为5500V和-4500V，雾化温度为650℃，雾化气（GS1，N_2）为60psi，辅助气（GS2，N_2）为65psi，气帘气（N_2）为25psi。
结果：13种活性成分的线性关系良好（r~2≥0.9941），检测限和定量限分别小于3.96ng/mL和9.90ng/mL，日内日间精密度分别小于2.86%和3.67%。平均回收率的范围为95.0%～104.4%，48h内的稳定性良好。并且采用ESI-MS/MS分析技术，对大蓟中12个黄酮类成分对照品的质谱裂解碎片进行分析，总结了黄酮类化合物的质谱裂解规律。
结论：本法简便、快速、灵敏度高、专属性好，可用于大蓟药材中柳穿鱼叶苷、柳穿鱼黄素、蒙花苷、刺槐素、芦丁、香叶木素、高车前素、芹菜素、柚皮素、橙皮苷、木犀草素、槲皮素和原儿茶酸的含量测定，为大蓟药材的质量控制提供了新的方法和手段。首次总结了大蓟中黄酮类成分ESI-MS/MS裂解规律。
Cirsium japonicum and Cirsium setosum, the members of the familyCompositae, is a wild perennial herb found in many areas of China. They arethe famous traditional Chinese medicine (TCM) and have been used forthousands of years to treat diverse kinds of bleeding (e.g. haematuria, spittingof blood, uterine bleeding) and inflammation. Modern pharmacologicalstudies demonstrated that Cirsium japonicum and Cirsium setosum extractshad a number of bioactivities, including anthemorrhagic, anti-inflammatory,anticancer and antimicrobial activities. Phytochemical studies on Cirsiumjaponicum and Cirsium setosum revealed that they both contained flavonoids,phenolic acids, sterols, triterpenes, alkaloids and so on. Among these,flavonoids and phenolic acids are generally considered to be the major activecomponents.
To date, studies on quantitative determination of chemical constituents inCirsium japonicum and Cirsium setosum and their pharmacokinetics havebeen very few. In present study, we firstly developed an accurate and simpleLC-ESI-MS/MS method for simultaneous determination of the majorcomponents in Cirsium japonicum and Cirsium setosum. The satisfactoryresults demonstrated that the LC-ESI-MS/MS method was a good option forroutine analysis and could be applied as a reliable quality control method forCirsium japonicum and Cirsium setosum. In this study, we developed a rathersensitive and selective LC-ESI-MS/MS method to simultaneously determinelinarin, acacetin, rutin, hesperidin, luteolin, apigenin and protocatechuic acidin rat plasma. The method was applied to pharmacokinetics after oraladministration of Cirsium setosum extract to rats. A sensitive and selectiveLC-ESI-MS/MS method was developed for simultaneous determination of the 7main active components in rat bile and urine and was applied to theexcretion amount study of the analytes after oral administration of Cirsiumsetosum extract. The obtained results would be very helpful for evaluating theclinical application of this herb.
Part one Simultaneous analysis of11main active components inCirsium setosum based on LC-ESI-MS/MS and combinedwith statistical methods
Objective: A novel method based on high-performance liquidchromatography coupled with electrospray ionization tandem massspectrometry was developed for simultaneous determination of the11majoractive components including10flavonoids (linarin, acacetin, rutin, diosmetin,hispidulin, apigenin, naringenin, hesperidin, luteolin and quercetin) and1phenolic acid (protocatechuic acid) in Cirsium setosum. The principalcomponent analysis (PCA) and hierarchical cluster analysis (HCA) wereemployed to classify and evaluate the25batches of Cirsium setosum samplesfrom different resources.
Methods: Chromatographic separation was performed on a C_(18)columnwith linear gradient elution of methanol and0.1‰acetic acid (v/v) at a flowrate of0.8mL/min. The total run time was19min. Multiple-reactionmonitoring (MRM) was employed in positive and negative mode at the sametime in single analysis process. The operating conditions were as follows: theion spray voltage was set to5500and-4500V, respectively; the turbo spraytemperature was650℃; nitrogen was used as the nebulizer gas (60psi) andheater gas (65psi); the curtain gas was kept at25psi and interface heater wason. The effect of origin in Cirsium setosum on the total amount of thoseanalytes was analyzed by PCA using SPSS. The HCA of Samples1-25wasperformed using SPSS software.
Results: The correlation coefficients were all higher than0.9941. TheLODs and LOQs for each compound were less than3.96ng/mL and9.90ng/mL, which showed a high sensitivity. The overall intra-and inter-dayprecisions (RSD) for the investigated components were less than3.30%and 3.57%, respectively. The average recovery was in the range of96.4%-104.2%.All analytes were found to be stable with48h. The results demonstrated thatthe quantitative difference of eleven active compounds was useful forchemotaxonomy of many samples from different sources and thestandardization and differentiation of many similar samples. Principalcomponent analysis and hierarchical clustering analysis were performed todifferentiate and classify the25batches of Cirsium setosum samples andfurther confirmed the excellent quality of Cirsium setosum from Hebeiprovince.
Conclusion: A efficient, rapid and sensitive LC-ESI-MS/MS methodoperating both positive and negative scanning modes in single analysisprocess was first established for the qualitation and quantification of11majorcomponents in Cirsium setosum. Validation of the assay showed appropriatesensitivity and specificity and was successfully utilized to analyze25batchesof Cirsium setosum samples from different sources. PCA and HCA validatedeach other and provided more evidence for the quality evaluation of Cirsiumsetosum samples. The satisfactory results demonstrated that theLC-ESI-MS/MS method was a good option for routine analysis and could beapplied as a reliable quality control method for Cirsium setosum. In the future,LC-ESI-MS/MS method will be more and more popular for analysis of herbalmedicine.
Part two Simultaneous determination and pharmacokinetic study ofseven main active components from Cirsium setosum extractin rat plasma by LC-ESI-MS/MS
Objective: To establish a sensitive, specific and rapid liquidchromatography-mass spectrometry (LC-ESI-MS/MS) method todetermination the seven main active components including linarin, acacetin,rutin, hesperidin, luteolin, apigenin and protocatechuic acid in rat plasma afterthe orally administrating of Cirsium setosum extract, and this method wasused and validated to study the pharmacokinetics.
Methods: Six rats were given single doses of Cirsium setosum extract (8 mL/kg) and blood samples were collected into heparinized centrifuge tubesfrom the vein of the eye ground0.17,0.5,1,1.5,2,3,4,6,8,12,24and36hafter a single oral administration. Within30min after blood withdrawal, thesamples were centrifuged at4000rpm for10min and the separated plasmasamples were frozen in polypropylene tubes at-20°C prior to analysis. Theplasma samples were pretreated and extracted by a simple liquid–liquidextraction (LLE) method by ethyl acetate. Sulfamethoxazole (SMZ) was usedas internal standard. Chromatographic conditions: Reverse-phase DiamonsilC_(18)column (150×4.6mm,5μm) with the column temperature set at30℃. Alinear gradient elution of eluents A (methanol) and B (0.1‰acetic acid; v/v)was used for the separation. The following gradient condition was used: initial0–1.5min, linear change from35%A to60%A;1.5–10min, linear changefrom60%A to63%A;10–10.1min, linear change from63%A to95%A; and10.1–15min, isocratic elution95%A; finally35%A maintained for6min. Theflow rate was set at0.8mL/min. Mass spectrometry: The mass spectrometerwas operated by switching the ESI source between the positive and negativemodes for a single run. The ion spray voltage was set to5500V and4500V,the turbo spray temperature was kept at650℃. Nebulizer gas (gas1) andheater gas (gas2) was set at60and65arbitrary units, respectively. The curtaingas was kept at25arbitrary units. For structural identification of each analyte,the information-dependent acquisition (IDA) method was used to trigger theenhanced product ion (EPI) scans by analyzing MRM signals. The optimizedmass transition ion-pairs (m/z) for quantitation were593.3/285.3for linarin,283.1/267.9for acacetin,609.1/299.9for rutin,609.3/300.9for hesperidin,284.9/133.1for luteolin,269.0/117.0for apigenin,153.0/108.9forprotocatechuic acid and252.0/156.0for IS. The total run time was15.0minbetween injections.
Results: The calibration curves were linear over the investigatedconcentration range:1.87～935ng/mL (linarin),2.63～1315ng/mL (acacetin),7.80～3900ng/mL (rutin),2.68～1340ng/mL (hesperidin),1.99～995ng/mL(luteolin),1.53～765ng/mL (apigenin) and2.84～1420ng/mL (protocatechuic acid), with all correlation coefficients higher than0.9954. Thelower limits of quantitation (LLOQ) of these analytes were less than7.80ng/mL. The intra-and inter-day RSD were no more than9.4%and the relativeerrors were within the range of-4.8%to9.8%. The average extractionrecoveries for all compounds were between71.0%and89.7%. Linarin andprotocatechuic acid could achieve the maximum plasma concentration at2h,while rutin, hesperidin, luteolin and apigenin could achieve the maximumplasma concentration at3h after oral administration. But acacetin need6h toachieve the maximum plasma concentration after oral administration Theseven analytes have distinctive pharmacokinetic parameters in vivo. All of theseven analytes were absorbed rapidly and have the similar elimination rate.
Conclusion: A selective LC-ESI-MS/MS method was developed andvalidated for the simultaneous determination of linarin, acacetin, rutin,hesperidin, luteolin, apigenin and protocatechuic acid in rat plasma after theorally administrating of Cirsium setosum extract. The results showed that thismethod is robust, specific and sensitive and it can successfully fulfill therequirements of pharmacokinetic study. The results provided a meaningfulbasis for the clinical application of this herb.
Part three Simultaneous determination and excretion study of sevenmain active components in rat bile and urine after oraladministration of Cirsium setosum extract byLC/electrospray ionization mass spectrometry
Objective: A sensitive and selective LC-ESI-MS/MS method wasdeveloped and validated for simultaneous analysis of seven main activecomponents (linarin, acacetin, rutin, hesperidin, luteolin, apigenin andprotocatechuic acid) in rat bile and urine. Then, the excretion profiles of thesecomponents were further investigated after a single oral administration ofCirsium setosum extract.
Methods: Six rats were administered with Cirsium setosum extract at asingle oral dosage of8mL/kg. Bile samples were collected during0-2,2-4,4-6,6-8,8-12,12-24,24-30,30-36h periods. Urine samples were collected during0-4,4-8,8-12,12-24,24-36,36-48,48-60,60-72,72-84h periods.Blank bile and urine samples were collected to check whether they were freeof interfering components. All samples were stored at-20℃until additionalextraction and analysis. The seven main active components and IS wereextracted by a simple liquid–liquid extraction (LLE) method by ethyl acetate.Sulfamethoxazole (SMZ) was used as internal standard. Chromatographicconditions: Reverse-phase Diamonsil C_(18)column (150×4.6mm,5μm) withthe column temperature set at30℃. A linear gradient elution of eluents A(methanol) and B (0.1‰acetic acid; v/v) was used for the separation. Thefollowing gradient condition was used: initial0–1.5min, linear change from35%A to60%A;1.5–10min, linear change from60%A to63%A;10–10.1min, linear change from63%A to95%A; and10.1–15min, isocratic elution95%A; finally35%A maintained for6min. The flow rate was set at0.8mL/min. Mass spectrometry: The mass spectrometer was operated byswitching the ESI source between the positive and negative modes for a singlerun. The ion spray voltage was set to5500V and4500V, the turbo spraytemperature was kept at650℃. Nebulizer gas (gas1) and heater gas (gas2)was set at60and65arbitrary units, respectively. The curtain gas was kept at25arbitrary units. For structural identification of each analyte, theinformation-dependent acquisition (IDA) method was used to trigger theenhanced product ion (EPI) scans by analyzing MRM signals. The optimizedmass transition ion-pairs (m/z) for quantitation were593.3/285.3for linarin,283.1/267.9for acacetin,609.1/299.9for rutin,609.3/300.9for hesperidin,284.9/133.1for luteolin,269.0/117.0for apigenin,153.0/108.9forprotocatechuic acid and252.0/156.0for IS. The total run time was15.0minbetween injections.
Results: The correlation coefficients were all higher than0.9930. Theresults of the inter-and intra-day precision (≤9.6%) and accuracy (within±15%) at QC concentrations were acceptable. The extraction recovery ofanalytes ranged from65.0%to81.2%for bile, and from71.0%to83.6%forurine. For matrix effect, the values ranged from85.4%to104.6%for bile, and from92.0%to104.7%for urine, which indicates no matrix effect forquantification of the target flavones in the developed method. Stability dataindicated good stability for all the analytes over four storage conditions in bileand urine. In the bile samples, the six analytes (linarin, acacetin, rutin,hesperidin, apigenin and protocatechuic acid) excreted completely in thirty-sixhours and the cumulative biliary excretion of six analytes excreted were0.499%,0.088%,0.163%,0.283%,0.146%,0.338%, respectively. In the urinesamples, the five analytes (linarin, acacetin, hesperidin, luteolin and apigenin)excreted completely in eighty-four hours. The cumulative urinary excretion offive analytes excreted were0.015%,1.164%,0.329%,0.201%,2.182%,respectively.
Conclusion: The method is robust and specific and it can successfullycomplete the requirements of the excretion study of seven main activecomponents in rat bile and urine in Cirsium setosum. These results may offeruseful information for clinical application of traditional Chinese medicines.
Part four Simultaneous analysis of13main active components inCirsium joponicum based on LC-ESI-MS/MS andfragmentation study of flavonoids with ESI-MS/MS innegative ion mode
Objective: A novel method based on high-performance liquidchromatography coupled with electrospray ionization tandem massspectrometry was developed for simultaneous determination of the13majoractive components including12flavonoids (pectolinarin, pectolinarigenin,linarin, acacetin, rutin, diosmetin, hispidulin, apigenin, naringenin, hesperidin,luteolin and quercetin) and1phenolic acid (protocatechuic acid) in Cirsiumjoponicum.12flavonoids were selected to research mass spectrum applicationwith ESI-MS/MS and the fragmentation rules of flavonoids profits for theidentification of unknown flavonoid compounds rapidly and accurately.
Methods: Chromatographic separation was performed on a C_(18)columnwith linear gradient elution of methanol and0.1‰acetic acid (v/v) at a flowrate of0.8mL/min. The total run time was19min. Multiple-reaction monitoring (MRM) was employed in positive and negative mode at the sametime in single analysis process. The operating conditions were as follows: theion spray voltage was set to5500and-4500V, respectively; the turbo spraytemperature was650℃; nitrogen was used as the nebulizer gas (60psi) andheater gas (65psi); the curtain gas was kept at25psi and interface heater wason. The effect of origin in Cirsium joponicum on the total amount of thoseanalytes was analyzed by PCA using SPSS. The HCA of Samples1-25wasperformed using SPSS software.
Results: The correlation coefficients were all higher than0.9941. TheLODs and LOQs for each compound were less than3.96ng/mL and9.90ng/mL, which showed a high sensitivity. The overall intra-and inter-dayprecisions (RSD) for the investigated components were less than2.86%and3.67%, respectively. The average recovery was in the range of95.0%-104.4%.All analytes were found to be stable with48h. Four fragmentation rules offlavonoids were summarized.
Conclusion: A efficient, rapid and sensitive LC-ESI-MS/MS methodoperating both positive and negative scanning modes in single analysisprocess was first established for the qualitation and quantification of11majorcomponents in Cirsium joponicum. Validation of the assay showedappropriate sensitivity and specificity and was successfully utilized to analyze25batches of Cirsium joponicum samples from different sources. Thesatisfactory results demonstrated that the LC-ESI-MS/MS method was a goodoption for routine analysis and could be applied as a reliable quality controlmethod for Cirsium joponicum. In the future, LC-ESI-MS/MS method will bemore and more popular for analysis of herbal medicine. Fragmentation rulesof12flavonoids in Cirsium joponicum were first study.
迄今为止国内外学者对大小蓟的研究主要集中在化学成分鉴定与分离,而对于其质量评价与体内药代动力学研究涉及较少。本文采用LC-ESI-MS/MS技术对大小蓟中的黄酮和酚酸类成分同时进行定性及定量研究，为中药材大蓟和小蓟的质量控制提供新的分析手段。且研究了小蓟中7种活性成分在动物体内的动力学过程和排泄规律，对中药材小蓟的临床应用提供依据。
第一部分液质联用法同时测定小蓟中11种活性成分的含量及化学计量学分析
目的：建立一种快速、准确的LC-ESI-MS/MS定量分析方法，可同时分离、测定小蓟中11种活性成分（10种黄酮，包括蒙花苷、刺槐素、芦丁、香叶木素、高车前素、芹菜素、柚皮素、橙皮苷、木犀草素和槲皮素；1种酚酸，原儿茶酸），并用于小蓟药材的质量分析。采用主成分分析及聚类分析的统计方法分类和区分25批不同产地的小蓟药材。
方法：液相色谱柱为Diamonsil C_(18)柱（150×4.6mm,5μm），流动相为甲醇：水（含有0.1‰乙酸），流速为0.8mL/min，分析时间为19min，梯度洗脱。质谱采用电喷雾离子源（ESI源），TurboV离子源，采用多反应离子监测（MRM），进行正负离子同时检测。源喷射电压（IS）为5500V和-4500V，雾化温度为650℃，雾化气（GS1，N_2）为60psi，辅助气（GS2，N_2）为65psi，气帘气（N_2）为25psi。实验采用SPSS统计软件对数据进行统计分析。
结果：11种活性成分的线性关系良好（r~2≥0.9941），检测限和定量限分别小于3.96ng/mL和9.90ng/mL，日内日间精密度分别小于3.30%和3.57%。平均回收率范围在96.4%～104.2%，48h内的稳定性良好。主成分分析及聚类分析对25批小蓟药材成功分类区分，证明河北产药材质量最佳。
结论：本法简便、快速、灵敏度高、专属性好，可用于小蓟药材中蒙花苷、刺槐素、芦丁、香叶木素、高车前素、芹菜素、柚皮素、橙皮苷、木犀草素、槲皮素和原儿茶酸的含量测定，为小蓟药材的质量控制提供了新的方法和手段。
第二部分液质联用法同时测定大鼠口服小蓟提取物后血浆中7种成分及药代动力学研究
目的：建立一种同时测定大鼠血浆中蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸含量的LC-ESI-MS/MS方法，并成功的用于大鼠灌胃给予小蓟提取物后血浆中7种成分的药代动力学研究，并建立其药-时曲线，阐明药动学参数与特征。
方法：大鼠灌胃给与小蓟提取物（8mL/kg）后，分别于给药后0.17，0.5，1，1.5，2，3，4，6，8，12，24和36h眼内眦静脉丛取血0.3mL，制备血浆样品，采用乙酸乙酯液-液提取方法进行样品预处理，磺胺甲噁唑为内标。反相Diamonsil C_(18)柱（150×4.6mm,5μm），流动相为A（甲醇）-B（0.1‰乙酸水），梯度洗脱：0.0-1.5min，35%-60%A；1.5-10.0min，60%-63%A；10.0-10.1min，63%-95%A；10.1-15.0min，95%A等度洗脱。进样前预平衡6min，柱温为30℃，流速为0.8mL/min。质谱条件：ESI源，源电压（IS）5500V和-4500V，源温度（TEM）650℃，雾化气（GS1，N_2）60psi，辅助气（GS2，N_2）65psi，气帘气（N_2）25psi。采用电喷雾离子源（ESI），多周期正负离子同时扫描，多反应监测（MRM）模式进行定量。7种被测成分和内标物的监测离子对分别为蒙花苷m/z593.3/285.3、刺槐素m/z283.1/267.9、芦丁m/z609.1/299.9、橙皮苷m/z609.3/300.9、木犀草素m/z284.9/133.1、芹菜素m/z269.0/117.0、原儿茶酸m/z153.0/108.9和磺胺甲噁唑m/z252.0/156.0。
结果：血浆中蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸分别在1.87～935、2.63～1315、7.80～3900、2.68～1340、1.99～995、1.53～765和2.84～1420ng/mL范围内线性关系良好（r~2≥0.9954），最低定量限（LLOQ）≤7.80ng/mL。日内、日间精密度的相对标准偏差（RSD）均小于9.4%，相对误差（RE）为-4.8%～9.8%。平均提取回收率为71.0%～89.7%。大鼠口服灌胃给予小蓟提取物后，血浆中7种待测成分蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸在大鼠体内的药代动力学药-时曲线的有所差异。其中，蒙花苷和原儿茶酸吸收最快，达峰时间为给药后2h左右；芦丁、橙皮苷、木犀草素和芹菜素吸收稍慢，达峰时间为给药后3h左右；刺槐素吸收最慢，于给药后6h达到血浆峰浓度。总体来说，大鼠灌胃给予小蓟提取物后，7种待测成分在体内吸收均较迅速，10min均可检测到，并且具有相近的消除速率。
结论：本研究建立了LC-ESI-MS/MS法同时测定大鼠血浆中蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸的含量，可用于小蓟中7种活性成分在大鼠体内的药代动力学研究。该方法简便、快速、灵敏度高、专属性好，对小蓟药材的体内定量分析具有重要意义，有益于传统中药小蓟的临床应用。
第三部分液质联用法同时测定大鼠口服小蓟提取物后胆汁和尿液中7种成分及排泄动力学研究
目的：建立LC-ESI-MS/MS同时测定大鼠胆汁和尿液中7种成分（蒙花苷、刺槐素、芦丁、橙皮苷、木犀草素、芹菜素和原儿茶酸）含量的方法，并用于大鼠灌胃给予小蓟提取物后该7种成分的胆汁和尿液排泄动力学研究。
方法：大鼠灌胃给予小蓟提取物8mL/kg，胆汁按照0-2，2-4，4-6，6-8，8-12，12-24，24-30，30-36h收集，尿液按照0-4，4-8，8-12，12-24，24-36，36-48，48-60，60-72，72-84h时间段收集。采用乙酸乙酯液-液提取方法进行样品前处理，磺胺甲噁唑为内标。反相Diamonsil C_(18)柱（150×4.6mm,5μm），流动相为A（甲醇）-B（0.1‰乙酸水），梯度洗脱：0.0-1.5min，35%-60%A；1.5-10.0min，60%-63%A；10.0-10.1min，63%-95%A；10.1-15.0min，95%A等度洗脱。进样前预平衡6min，柱温为30℃，流速为0.8mL/min。质谱条件：ESI源，源电压（IS）5500V和-4500V，源温度（TEM）650℃，雾化气（GS1，N_2）60psi，辅助气（GS2，N_2）65psi，气帘气（N_2）25psi。采用电喷雾离子源（ESI），多周期正负离子同时扫描，多反应监测（MRM）模式进行定量。7种被测成分和内标物的监测离子对分别为蒙花苷m/z593.3/285.3、刺槐素m/z283.1/267.9、芦丁m/z609.1/299.9、橙皮苷m/z609.3/300.9、木犀草素m/z284.9/133.1、芹菜素m/z269.0/117.0、原儿茶酸m/z153.0/108.9和磺胺甲噁唑m/z252.0/156.0。测定大鼠灌胃给予小蓟提取物后胆汁和尿液中7种成分的累积排泄率。
结果：胆汁和尿液中7种活性成分在测定浓度范围内均具有良好的线性关系（r~2≥0.9930），日内日间精密度均小于9.6%，相对误差RE均在±15%内。在胆汁样品中提取回收率为65.0%～81.2%，基质效应为85.4%～104.6%。尿液样品中提取回收率为71.0%～83.6%，基质效应为92.0%～104.7%。胆汁及尿液样品稳定性良好。试验结果表明，大鼠的胆汁中检测到蒙花苷、刺槐素、芦丁、橙皮苷、芹菜素和原儿茶酸6个分析物，它们的累计排泄率分别为0.499，0.088，0.163，0.283，0.146和0.338%，6个分析物36h基本排泄完全；大鼠的尿液中检测到蒙花苷、刺槐素、橙皮苷、木犀草素和芹菜素5个分析物，其累积排泄率分别为0.015，1.164，0.329，0.201和2.182%，5个分析物84h基本排泄完全。。
结论：本研究首次采用LC-ESI-MS/MS法测定了大鼠胆汁和尿液中小蓟中7种活性成分，并研究了其排泄动力学。方法准确，结果可靠，可满足中药材在生物基质中的排泄研究。该法对小蓟药材的进一步临床应用具有重要意义。
第四部分液质联用技术同时测定大蓟中13种成分及黄酮类成分的裂解规律分析
目的：建立一种快速、准确的LC-ESI-MS/MS定量分析方法，可同时分离、测定大蓟中13种成分（12种黄酮，包括柳穿鱼叶苷、柳穿鱼黄素、蒙花苷、刺槐素、芦丁、香叶木素、高车前素、芹菜素、柚皮素、橙皮苷、木犀草素和槲皮素；1种酚酸，原儿茶酸），并用于大蓟药材分析。采用ESI-MS/MS法推断大蓟药材中12种黄酮类化合物的裂解规律。
方法：液相色谱柱为Diamonsil C_(18)柱（150×4.6mm,5μm），流动相为甲醇：水（含有0.1‰乙酸），流速为0.8mL/min，分析时间为19min，梯度洗脱。质谱采用电喷雾离子源（ESI源），TurboV离子源，采用多反应离子监测（MRM），进行正负离子同时检测。源喷射电压（IS）为5500V和-4500V，雾化温度为650℃，雾化气（GS1，N_2）为60psi，辅助气（GS2，N_2）为65psi，气帘气（N_2）为25psi。
结果：13种活性成分的线性关系良好（r~2≥0.9941），检测限和定量限分别小于3.96ng/mL和9.90ng/mL，日内日间精密度分别小于2.86%和3.67%。平均回收率的范围为95.0%～104.4%，48h内的稳定性良好。并且采用ESI-MS/MS分析技术，对大蓟中12个黄酮类成分对照品的质谱裂解碎片进行分析，总结了黄酮类化合物的质谱裂解规律。
结论：本法简便、快速、灵敏度高、专属性好，可用于大蓟药材中柳穿鱼叶苷、柳穿鱼黄素、蒙花苷、刺槐素、芦丁、香叶木素、高车前素、芹菜素、柚皮素、橙皮苷、木犀草素、槲皮素和原儿茶酸的含量测定，为大蓟药材的质量控制提供了新的方法和手段。首次总结了大蓟中黄酮类成分ESI-MS/MS裂解规律。
Cirsium japonicum and Cirsium setosum, the members of the familyCompositae, is a wild perennial herb found in many areas of China. They arethe famous traditional Chinese medicine (TCM) and have been used forthousands of years to treat diverse kinds of bleeding (e.g. haematuria, spittingof blood, uterine bleeding) and inflammation. Modern pharmacologicalstudies demonstrated that Cirsium japonicum and Cirsium setosum extractshad a number of bioactivities, including anthemorrhagic, anti-inflammatory,anticancer and antimicrobial activities. Phytochemical studies on Cirsiumjaponicum and Cirsium setosum revealed that they both contained flavonoids,phenolic acids, sterols, triterpenes, alkaloids and so on. Among these,flavonoids and phenolic acids are generally considered to be the major activecomponents.
To date, studies on quantitative determination of chemical constituents inCirsium japonicum and Cirsium setosum and their pharmacokinetics havebeen very few. In present study, we firstly developed an accurate and simpleLC-ESI-MS/MS method for simultaneous determination of the majorcomponents in Cirsium japonicum and Cirsium setosum. The satisfactoryresults demonstrated that the LC-ESI-MS/MS method was a good option forroutine analysis and could be applied as a reliable quality control method forCirsium japonicum and Cirsium setosum. In this study, we developed a rathersensitive and selective LC-ESI-MS/MS method to simultaneously determinelinarin, acacetin, rutin, hesperidin, luteolin, apigenin and protocatechuic acidin rat plasma. The method was applied to pharmacokinetics after oraladministration of Cirsium setosum extract to rats. A sensitive and selectiveLC-ESI-MS/MS method was developed for simultaneous determination of the 7main active components in rat bile and urine and was applied to theexcretion amount study of the analytes after oral administration of Cirsiumsetosum extract. The obtained results would be very helpful for evaluating theclinical application of this herb.
Part one Simultaneous analysis of11main active components inCirsium setosum based on LC-ESI-MS/MS and combinedwith statistical methods
Objective: A novel method based on high-performance liquidchromatography coupled with electrospray ionization tandem massspectrometry was developed for simultaneous determination of the11majoractive components including10flavonoids (linarin, acacetin, rutin, diosmetin,hispidulin, apigenin, naringenin, hesperidin, luteolin and quercetin) and1phenolic acid (protocatechuic acid) in Cirsium setosum. The principalcomponent analysis (PCA) and hierarchical cluster analysis (HCA) wereemployed to classify and evaluate the25batches of Cirsium setosum samplesfrom different resources.
Methods: Chromatographic separation was performed on a C_(18)columnwith linear gradient elution of methanol and0.1‰acetic acid (v/v) at a flowrate of0.8mL/min. The total run time was19min. Multiple-reactionmonitoring (MRM) was employed in positive and negative mode at the sametime in single analysis process. The operating conditions were as follows: theion spray voltage was set to5500and-4500V, respectively; the turbo spraytemperature was650℃; nitrogen was used as the nebulizer gas (60psi) andheater gas (65psi); the curtain gas was kept at25psi and interface heater wason. The effect of origin in Cirsium setosum on the total amount of thoseanalytes was analyzed by PCA using SPSS. The HCA of Samples1-25wasperformed using SPSS software.
Results: The correlation coefficients were all higher than0.9941. TheLODs and LOQs for each compound were less than3.96ng/mL and9.90ng/mL, which showed a high sensitivity. The overall intra-and inter-dayprecisions (RSD) for the investigated components were less than3.30%and 3.57%, respectively. The average recovery was in the range of96.4%-104.2%.All analytes were found to be stable with48h. The results demonstrated thatthe quantitative difference of eleven active compounds was useful forchemotaxonomy of many samples from different sources and thestandardization and differentiation of many similar samples. Principalcomponent analysis and hierarchical clustering analysis were performed todifferentiate and classify the25batches of Cirsium setosum samples andfurther confirmed the excellent quality of Cirsium setosum from Hebeiprovince.
Conclusion: A efficient, rapid and sensitive LC-ESI-MS/MS methodoperating both positive and negative scanning modes in single analysisprocess was first established for the qualitation and quantification of11majorcomponents in Cirsium setosum. Validation of the assay showed appropriatesensitivity and specificity and was successfully utilized to analyze25batchesof Cirsium setosum samples from different sources. PCA and HCA validatedeach other and provided more evidence for the quality evaluation of Cirsiumsetosum samples. The satisfactory results demonstrated that theLC-ESI-MS/MS method was a good option for routine analysis and could beapplied as a reliable quality control method for Cirsium setosum. In the future,LC-ESI-MS/MS method will be more and more popular for analysis of herbalmedicine.
Part two Simultaneous determination and pharmacokinetic study ofseven main active components from Cirsium setosum extractin rat plasma by LC-ESI-MS/MS
Objective: To establish a sensitive, specific and rapid liquidchromatography-mass spectrometry (LC-ESI-MS/MS) method todetermination the seven main active components including linarin, acacetin,rutin, hesperidin, luteolin, apigenin and protocatechuic acid in rat plasma afterthe orally administrating of Cirsium setosum extract, and this method wasused and validated to study the pharmacokinetics.
Methods: Six rats were given single doses of Cirsium setosum extract (8 mL/kg) and blood samples were collected into heparinized centrifuge tubesfrom the vein of the eye ground0.17,0.5,1,1.5,2,3,4,6,8,12,24and36hafter a single oral administration. Within30min after blood withdrawal, thesamples were centrifuged at4000rpm for10min and the separated plasmasamples were frozen in polypropylene tubes at-20°C prior to analysis. Theplasma samples were pretreated and extracted by a simple liquid–liquidextraction (LLE) method by ethyl acetate. Sulfamethoxazole (SMZ) was usedas internal standard. Chromatographic conditions: Reverse-phase DiamonsilC_(18)column (150×4.6mm,5μm) with the column temperature set at30℃. Alinear gradient elution of eluents A (methanol) and B (0.1‰acetic acid; v/v)was used for the separation. The following gradient condition was used: initial0–1.5min, linear change from35%A to60%A;1.5–10min, linear changefrom60%A to63%A;10–10.1min, linear change from63%A to95%A; and10.1–15min, isocratic elution95%A; finally35%A maintained for6min. Theflow rate was set at0.8mL/min. Mass spectrometry: The mass spectrometerwas operated by switching the ESI source between the positive and negativemodes for a single run. The ion spray voltage was set to5500V and4500V,the turbo spray temperature was kept at650℃. Nebulizer gas (gas1) andheater gas (gas2) was set at60and65arbitrary units, respectively. The curtaingas was kept at25arbitrary units. For structural identification of each analyte,the information-dependent acquisition (IDA) method was used to trigger theenhanced product ion (EPI) scans by analyzing MRM signals. The optimizedmass transition ion-pairs (m/z) for quantitation were593.3/285.3for linarin,283.1/267.9for acacetin,609.1/299.9for rutin,609.3/300.9for hesperidin,284.9/133.1for luteolin,269.0/117.0for apigenin,153.0/108.9forprotocatechuic acid and252.0/156.0for IS. The total run time was15.0minbetween injections.
Results: The calibration curves were linear over the investigatedconcentration range:1.87～935ng/mL (linarin),2.63～1315ng/mL (acacetin),7.80～3900ng/mL (rutin),2.68～1340ng/mL (hesperidin),1.99～995ng/mL(luteolin),1.53～765ng/mL (apigenin) and2.84～1420ng/mL (protocatechuic acid), with all correlation coefficients higher than0.9954. Thelower limits of quantitation (LLOQ) of these analytes were less than7.80ng/mL. The intra-and inter-day RSD were no more than9.4%and the relativeerrors were within the range of-4.8%to9.8%. The average extractionrecoveries for all compounds were between71.0%and89.7%. Linarin andprotocatechuic acid could achieve the maximum plasma concentration at2h,while rutin, hesperidin, luteolin and apigenin could achieve the maximumplasma concentration at3h after oral administration. But acacetin need6h toachieve the maximum plasma concentration after oral administration Theseven analytes have distinctive pharmacokinetic parameters in vivo. All of theseven analytes were absorbed rapidly and have the similar elimination rate.
Conclusion: A selective LC-ESI-MS/MS method was developed andvalidated for the simultaneous determination of linarin, acacetin, rutin,hesperidin, luteolin, apigenin and protocatechuic acid in rat plasma after theorally administrating of Cirsium setosum extract. The results showed that thismethod is robust, specific and sensitive and it can successfully fulfill therequirements of pharmacokinetic study. The results provided a meaningfulbasis for the clinical application of this herb.
Part three Simultaneous determination and excretion study of sevenmain active components in rat bile and urine after oraladministration of Cirsium setosum extract byLC/electrospray ionization mass spectrometry
Objective: A sensitive and selective LC-ESI-MS/MS method wasdeveloped and validated for simultaneous analysis of seven main activecomponents (linarin, acacetin, rutin, hesperidin, luteolin, apigenin andprotocatechuic acid) in rat bile and urine. Then, the excretion profiles of thesecomponents were further investigated after a single oral administration ofCirsium setosum extract.
Methods: Six rats were administered with Cirsium setosum extract at asingle oral dosage of8mL/kg. Bile samples were collected during0-2,2-4,4-6,6-8,8-12,12-24,24-30,30-36h periods. Urine samples were collected during0-4,4-8,8-12,12-24,24-36,36-48,48-60,60-72,72-84h periods.Blank bile and urine samples were collected to check whether they were freeof interfering components. All samples were stored at-20℃until additionalextraction and analysis. The seven main active components and IS wereextracted by a simple liquid–liquid extraction (LLE) method by ethyl acetate.Sulfamethoxazole (SMZ) was used as internal standard. Chromatographicconditions: Reverse-phase Diamonsil C_(18)column (150×4.6mm,5μm) withthe column temperature set at30℃. A linear gradient elution of eluents A(methanol) and B (0.1‰acetic acid; v/v) was used for the separation. Thefollowing gradient condition was used: initial0–1.5min, linear change from35%A to60%A;1.5–10min, linear change from60%A to63%A;10–10.1min, linear change from63%A to95%A; and10.1–15min, isocratic elution95%A; finally35%A maintained for6min. The flow rate was set at0.8mL/min. Mass spectrometry: The mass spectrometer was operated byswitching the ESI source between the positive and negative modes for a singlerun. The ion spray voltage was set to5500V and4500V, the turbo spraytemperature was kept at650℃. Nebulizer gas (gas1) and heater gas (gas2)was set at60and65arbitrary units, respectively. The curtain gas was kept at25arbitrary units. For structural identification of each analyte, theinformation-dependent acquisition (IDA) method was used to trigger theenhanced product ion (EPI) scans by analyzing MRM signals. The optimizedmass transition ion-pairs (m/z) for quantitation were593.3/285.3for linarin,283.1/267.9for acacetin,609.1/299.9for rutin,609.3/300.9for hesperidin,284.9/133.1for luteolin,269.0/117.0for apigenin,153.0/108.9forprotocatechuic acid and252.0/156.0for IS. The total run time was15.0minbetween injections.
Results: The correlation coefficients were all higher than0.9930. Theresults of the inter-and intra-day precision (≤9.6%) and accuracy (within±15%) at QC concentrations were acceptable. The extraction recovery ofanalytes ranged from65.0%to81.2%for bile, and from71.0%to83.6%forurine. For matrix effect, the values ranged from85.4%to104.6%for bile, and from92.0%to104.7%for urine, which indicates no matrix effect forquantification of the target flavones in the developed method. Stability dataindicated good stability for all the analytes over four storage conditions in bileand urine. In the bile samples, the six analytes (linarin, acacetin, rutin,hesperidin, apigenin and protocatechuic acid) excreted completely in thirty-sixhours and the cumulative biliary excretion of six analytes excreted were0.499%,0.088%,0.163%,0.283%,0.146%,0.338%, respectively. In the urinesamples, the five analytes (linarin, acacetin, hesperidin, luteolin and apigenin)excreted completely in eighty-four hours. The cumulative urinary excretion offive analytes excreted were0.015%,1.164%,0.329%,0.201%,2.182%,respectively.
Conclusion: The method is robust and specific and it can successfullycomplete the requirements of the excretion study of seven main activecomponents in rat bile and urine in Cirsium setosum. These results may offeruseful information for clinical application of traditional Chinese medicines.
Part four Simultaneous analysis of13main active components inCirsium joponicum based on LC-ESI-MS/MS andfragmentation study of flavonoids with ESI-MS/MS innegative ion mode
Objective: A novel method based on high-performance liquidchromatography coupled with electrospray ionization tandem massspectrometry was developed for simultaneous determination of the13majoractive components including12flavonoids (pectolinarin, pectolinarigenin,linarin, acacetin, rutin, diosmetin, hispidulin, apigenin, naringenin, hesperidin,luteolin and quercetin) and1phenolic acid (protocatechuic acid) in Cirsiumjoponicum.12flavonoids were selected to research mass spectrum applicationwith ESI-MS/MS and the fragmentation rules of flavonoids profits for theidentification of unknown flavonoid compounds rapidly and accurately.
Methods: Chromatographic separation was performed on a C_(18)columnwith linear gradient elution of methanol and0.1‰acetic acid (v/v) at a flowrate of0.8mL/min. The total run time was19min. Multiple-reaction monitoring (MRM) was employed in positive and negative mode at the sametime in single analysis process. The operating conditions were as follows: theion spray voltage was set to5500and-4500V, respectively; the turbo spraytemperature was650℃; nitrogen was used as the nebulizer gas (60psi) andheater gas (65psi); the curtain gas was kept at25psi and interface heater wason. The effect of origin in Cirsium joponicum on the total amount of thoseanalytes was analyzed by PCA using SPSS. The HCA of Samples1-25wasperformed using SPSS software.
Results: The correlation coefficients were all higher than0.9941. TheLODs and LOQs for each compound were less than3.96ng/mL and9.90ng/mL, which showed a high sensitivity. The overall intra-and inter-dayprecisions (RSD) for the investigated components were less than2.86%and3.67%, respectively. The average recovery was in the range of95.0%-104.4%.All analytes were found to be stable with48h. Four fragmentation rules offlavonoids were summarized.
Conclusion: A efficient, rapid and sensitive LC-ESI-MS/MS methodoperating both positive and negative scanning modes in single analysisprocess was first established for the qualitation and quantification of11majorcomponents in Cirsium joponicum. Validation of the assay showedappropriate sensitivity and specificity and was successfully utilized to analyze25batches of Cirsium joponicum samples from different sources. Thesatisfactory results demonstrated that the LC-ESI-MS/MS method was a goodoption for routine analysis and could be applied as a reliable quality controlmethod for Cirsium joponicum. In the future, LC-ESI-MS/MS method will bemore and more popular for analysis of herbal medicine. Fragmentation rulesof12flavonoids in Cirsium joponicum were first study.
引文
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4韩百翠，李宁，李铣.小蓟化学成分的分离与鉴定.沈阳药科大学学报，2008,25(10):793-795
5陈毓，丁安伟，杨星昊，等.小蓟化学成分药理作用及临床应用研究述要.中医药学刊，2005,23(4):614-615
6潘珂，尹永芹，孔令义.小蓟化学成分的研究.中国现代中药，2006,8(4):7-9
7许浚，张铁军，龚苏晓，等.小蓟止血活性部位的化学成分研究.中草药，2010,41(4):542-544
8周清，陈玲，刘志鹏，等.小蓟的化学成分研究.中药材，2007,30(1):45-47
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