| Oridonin(ORI)is an active natural ent-kaurane diterpenoid ingredient extracted from many plants belong to Isodon species.Recently,because of its strong anti-cancer and anti-tumor bioactivities,ORI is expected to develop into a novel anti-tumor and anti-cancer agent.Up to now,results in pharmacological and elimination studies indicated that ORI in vivo may occur significant biotransformation.Drug metabolism study is an important part in the development of new drugs.Therefore,it is necessary to study the metabolites of ORI.At the same time,it helps to offer early research data for the further pharmacology and toxicology study.In this study,ultra-high-performance liquid chromatography coupled with high-resolution hybrid quadrupole time-of flight mass spectrometer(UPLC-Q-TOF-MS/MS)was develoed to identify the metabolites of ORI in vivo.Simultaneously,the cleavage pathway of the possible metabolites was summaried and the possible metabolite pathway was deduced.Isodon japonica var.glaucocalyx(Maxim.)Hara is used as a well-known traditional medicinal herb.The whole plant and leaves of this herb has long been used as a folk medicine for hepatitis,gastritis,mastitis,tonsillitis,liver cancer and breast cancer.Isodon japonica var.glaucocalyx(Maxim.)Hara widely distributed in China.The Isodon plants were similar in appearance,so the market plant-based source was confused.At the same time,the contents of various chemical compounds in Isodon japonica var.glaucocalyx(Maxim.)Hara were influenced by many factors,such as geographical factors,the part of plants and different harvest time.Therefore,it is essential for simultaneous determination of major active chemical constituents for the quality control.Inthis study,high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)techniques was used to simultaneous determination of eight ent-kaurane diterpenoids in Isodon japonica var.glaucocalyx(Maxim.)Hara for the first time and was successly applied to analyze 31 batches samples.Principal component analysis(PCA)was introduced for better evaluate these samples quality.For comprehensive understanding those trace diterpenoids,the UPLC-Q-TOF-MS/MS was employed to rapidly identification and structural analysis of diterpenoid constituents.These results would provide foundation for depth understanding of the active ingredient and the effectiveness of traditional Chinese medicine.Part one Identification of metabolites of oridonin in rats urine and bile by UPLC-Q-TOF-MS/MSObjective: To develop HPLC-QTof-MS method for catching and indentifying of oridonin metabolites in rat urine and bile after oral administration,as well as to illustrate metabolites and pathways rule.Methods: Urine and bile samples were collected after single oral administration of oridonin to rats.Then the samples were pretreated by liquid-liquid extraction with ethyl acetate(twice amount of the samples v/v for3 times).A novel and efficient strategy was developed for screening and identification of oridonin metabolites in vivo using ultra high performance liquid chromatography combined with Triple TOF mass spectrometry(UPLC-TOF/MS/MS)with the negative ion electrospray mode in the scanning mode of TOF-MS-IDA-8MS/MS to acquire exact mass and MS/MS data sets of the unknown metabolites.Secondly,a combination of data mining methods including extracted ion chromatography(XIC),mass defects(MDF),product ion filte(PIF)and neutral loss filter(NLF)were performed to identify the metabolites.Accurate molecular mass,elemental composition and mass spectrum were obtained.Finally,the structures of the metabolites were elucidated based on the accurate mass measurement,relevant drug biotransformation knowledge and the fragmentation patterns of the parent drug.Furthermore,Clog P was used to distinguish isomers.Thechromatographic separation was carried on a Phenomenex Kinetex C18 column(100 mm×2.1 mm,2.6 ?m)with methanol and water containing 0.1% formic acid as gradient eluents.The temperature was set at 25oC.The mobile phase flow rate was set at 0.4 mL/min and the injection volume was 5 ?L.The elution programme was optimized for 15 min.Results: Based on the proposed strategy,18 metabolites(including 16 phase I and 2 phase II metabolites)were identified in rat after oral administration of ORI.Among them,10 metabolites in urine and 17 metabolites in bile were identified.The results indicated that ORI mainly underwent reduction and oxidative dehydrogenation reaction and that hydroxy group and carbon-carbon double bond were the metabolic sites.Conjugations reaction with glucuronic acid was the predominant phase-II metabolite reactions.Conclusion: This is the first report of the screening and identification of metabolites of ORI in vivo by UPLC-Q-TOF-MS/MS,which will be helpful to better understand the metabolism of ORI in vivo.This study provided a practical strategy for rapidly screening and identifying metabolites,and this methodology could be widely applied for the structural characterization of other compounds and TCMs metabolites.Part two Simultaneous quantification of 8 diterpenoids in Isodon japonica var.glaucocalyx(Maxim.)Hara by LC-MS/MS and principal component analysisObjective: To develop a high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)method for the simultaneous determination of 8 diterpenoids(hebeirubesensin K,enmenol,henryin,lasiodonin,glaucocalyxin A,oridonin,ponicidin and rabdoternin A)in Isodon japonica var.glaucocalyx(Maxim.)Hara.At the same time,principal component analysis(PCA)was used for better evaluate these samples quality.Methods: Detection was carried out by multiple-reaction monitoring(MRM)mode using electrospary ionization in the negative mode.The conditions of MS/MS detector were as follows: ion spray voltage,-4.5 k V;ionsource heater,650 °C;curtain gas(CUR),25 psi,the interface heater was turned on;gas 1,60 psi;gas 2,65 psi.Nitrogen was used in all cases.The optimized mass transition ion-pair(m/z)for quantification were 365.2/317.2for hebeirubesensin K,361.3/343.2 for ponicidin,365.2/347.2 for enmenol,363.2/197.0 for lasiodonin,391.2/58.9 for henryin,331.0/313.0 for glaucocalyxin A,363.2/345.1 for oridonin and 363.2/255.1 for rabdoternin A.The chromatographic separation was performed on an Agilent ZORBAX SB-C18 column(4.6 mm × 250 mm,5 ?m)and the column temperature set at25 °C.The analytes were eluted with a gradient system consisting of methanol and water.The solvent flow rate was kept at 0.8mL/min and the injection volume was 10?L.The established method was subsequently applied to analyze 31 samples from different areas and different parts.PCA was carried out for finding main factors related to quality.Results: The linear relationships,linearity range,repeatability precision,stability,LOD and LOQ of the method were good for the analyzed components.The average recoveries for the eight analytes were in the range of93.45–103.3% with RSD valuesof less than 5.6%.The results showed that the contents of eight chemical compositions were influenced by geographical factors,the part of plants and different harvest time.While the quantitative analytical results indicated that their contents of 8 major bioactive compounds varied in some degree.Glaucocalyxin A and henryin was found to be the predominant components.A two-component PCA score scatter plot showed that samples were successfully divided into two clusters.The results showed that the quality of herbal was influenced by growing locations.Conclusion: The method is quick,simple,rapid,specific,sensitive,accurate and reliable for the determination of multiple diterpenoids in Isodon japonica var.glaucocalyx(Maxim.)Hara.At the same time,principal component analysis can provide not only the basis for classification and quality evaluation but aslo the standard for the conservation and rational use of medicinal resources.Part three Rapid identification and qualitative analyses of diterpenoids in Isodon japonica var.glaucocalyx(Maxim.)Hara by UPLC-Q-TOF-MS/MS and MDF template technologyObjective: To establish a novel method based on ultra-high-performance liquid chromatography coupled with high-resolution hybrid quadrupole time-of flight mass spectrometer(UPLC-Q-TOF-MS)and MDF template technology for the rapid separation and identification of diterpenoids in Isodon japonica var.glaucocalyx(Maxim.)Hara.Methods: Firstly,the mass fragmentation and fragmentation patterns of eight major ent-kauranes diterpenoids were studied in the negative ion mode by full scan,product ion scan and precursor ion scan.Then the fragmentation rules were summarized.Analysis was performed on a UPLC-Q-TOF-MS/MS.Mass spectrometry was operating in the negative electrospray ionization(ESI)mode.The full MS-IDA(information dependent acquisition)-8MS/MS analysis,the eight most intense fragment ions of each analyte selected to do a production scan.Secondly,the unknown compound molecular weights and elemental compositions derived from the accurate mass measurements can be readily predicted,and accurate MS/MS spectra can also be interpreted to identify the diterpenoids of Isodon japonica var.glaucocalyx(Maxim.)Hara.The chromatographic separation was carried on a Phenomenex Kinetex C18(100 mm × 2.1 mm,2.6 ?m)with an on-line column filter.The column temperature was set at 25 °C.The mobile phase consisted of water(A)and methanol(B)with a flow rate of 0.3 mL/min.The injection volume was 2?L.Results: Four representative fragmentation rules were summarized by studying the mass fragmentation patterns of 8 standards in the negative ion mode by full scan,product ion scan and precursor ion scan.A total of 24ent-kaurane diterpenoids were identified or tentatively assigned based on this strategy.The known analytes were verified by comparing with the standards retention time and MS/MS information,and the other 16 compounds were identified by analyzing the information provided by XIC and MDF technique,the error tolerance is ±3 ppm.Conclusion: The developed UPLC-Q-TOF-MS/MS method is quick,simple,rapid,specific,sensitive,accurate and reliable for qualitative analysis of multiple trace diterpenoids in Isodon japonica var.glaucocalyx(Maxim.)Hara.This method provided a practical and feasible way to study of the chemical composition in TCM. |
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