| 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 methodsObjective: 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 C18columnwith 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/MSObjective: 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 DiamonsilC18column （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 spectrometryObjective: 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 C18column （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 modeObjective: 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 C18columnwith 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. |
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