| Anemarrhenae rhizoma (Zhimu in Chinese), the dried rhizome of A.asphodeloides Bunge (A. asphodeloides, Fam. Liliaceae), is a well-knowntraditional Chinese medicinal herb officially listed in the ChinesePharmacopoeia. In clinical applications of traditional Chinese medicine(TCM), this herb has proved effective in treating febrile diseases, high feverand thirst, heat in the lung with dry cough, osteopyrexia and fever, diabetesdue to internal heat, and constipation. Modern pharmacology studies show thatA. asphodeloides possesses antitumor, antiviral, immunomodulatory, andvascular modulatory activities, and can suppress the generation of superoxidein human neutrophils, exert remarkable inhibiting effects on plateletaggregation in human blood, and improve learning and memory abilities inmemory-deficit rat models. Phytochemical studies of Anemarrhenae haverevealed that it contains steroidal saponins, xanthone glycosides, isoflavonoids,as well as other chemicals. Some preparations which contains Anemarrhenaerhizoma have been used extensively in clinic.In this paper, HPLC-MS was performed to analyze chemical componentsin Anemarrhenae rhizoma extract and biological samples. Meanwhile, a novelsensitive and selective HPLC-ESI-MS/MS method was developed andvalidated to simultaneously determinate and identify constituents inAnemarrhenae rhizoma samples. Then, a sensitive and selective LC-ESI-MSmethod for the simultaneous determination of6analytes in rat plasma wasfirstly developed and validated to analyze plasma samples of the six analytesafter oral administration of the herbal preparation and the single herb extracts.Finally, a HPLC-MS method was established for the quantification of6analytes in rat bile and urine after oral administration of the herbal preparation and the single herb extracts.Part one Simultaneous determination of nine components inAnemarrhena by liquid chromatography-tandem massspectrometry combined with chemometric techniquesObjective: a novel quantitative method using high-performance liquidchromatography coupled to electrospray ionization tandemmassspectrometrywas developed for simultaneous determination of the importantactive constituents including four steroidal saponins, two xanthone glycosides,two isoflavonoids, and one anthraquinone in different parts of Anemarrhenaasphodeloides from different habitats. Hierarchical clustering analysis andprincipal components analysis were performed to differentiate and classify thesamples.Methods: The dry plant samples were ground to fine powder by apulverizer,and1.0g of powder was accurately weighed andultrasonicallyextracted with30mL of ethanol for1h. The extracted solutionwas adjusted to the original weight by adding ethanol, and then the aliquot ofthe supernatant was filtered through a0.45-mm microporous membrane beforeHPLC injection of10μL.The separation was performed on a C18column withacidified aqueous acetonitrile gradients. Quantification of the analytes wasachieved by use of a hybrid quadrupole linear ion-trap mass spectrometer.Multiple-reaction monitoring scanning was employed with switchingelectrospray ion source polarity between positive and negative modes in asingle run. The elution program was optimized as follows:0–2.5min, linearchange from A–B(5:95, v/v) to A–B (55:45, v/v);2.5–5min, linear changefrom A–B (55:45, v/v) to A–B (95:5, v/v); and5–20min, isocratic elutionA–B (95:5, v/v); then quickly returned to initial A–B (5:95, v/v). This wasfollowed by the equilibration period of6min prior to the injection of eachsample. The flow rate of mobile phase was set at0.8mL/min and the injectionvolume was10μL. The effect of processing method, origin, and different partsin A. asphodeloides on the total amount of the analytes was analyzed by HCAand PCA using SPSS software (SPSS Statistics19, SPSS Inc., USA). Results: As shown in Table3, the linear regression results indicate goodlinear correlation by the correlation coefficients of r2>0.9912for all thecompounds in the concentration range. The LOD and LOQ values of allcompounds are also summarized in Table3. It can be seen that the LOQ forthe components is much lower than that obtained in preceding studies[14], andsimilar to that of the results of in vivo studies[22,23], which indicates that theanalytical method was sufficiently sensitive. The overall intraday and interdayprecisions (RSD) for the investigated components were less than2.21and2.34%, respectively. The average recovery was in the range of96.8~103.9%with RSD ranging from0.81to2.32%. The results indicated that the method isaccurate and reproducible. These detailed results are summarized in Table4.When the solution was stored at4℃, all analytes were found to be stablewithin48h (RSD <2.28%). The proposed HPLC method was applied toanalyze the nine analytes in30batches of A. asphodeloides. The analysis timewas reduced to20min by switching the ion source polarity between positiveand negative modes in a single chromatographic run. Moreover, MRMscanning mode offered good sensitivity because it significantly decreased thenoise levels and accordingly enhanced the response of analytes. Thus, someminor constituents in A. asphodeloides could also be accurately measured. Thetarget compounds were identified on the basis of comparison of retention time,parent, and product obtained from LC-MS/MS analysis of the standardcompounds. The quantitative analysis was performed by means of the externalstandard methods. The data are summarized in Table5. It is found that thetotal contents of30batches of samples range from23.01to132.65mg/g,which indicates that A. asphodeloides samples were obviously different. HB-4,HB-5, and HB-6had the highest total contents (132.65,120.59, and116.92mg/g), while HBL-1, HBL-2, and HBL-3had the lowest total contents (23.01,34.09, and29.68mg/g). The mean value of the total content of30batches ofsamples was90.46mg/g. Six samples (HBP-1,-2,-3and HBS-1,-2,-3) had alittle lower content than the total mean content and HBL-1,-2,-3hadobviously lower content. Timosaponin BII was the component with the highest mean content of43.79mg/g, followed by mangiferin at36.13mg/g. Bothmethods of nearest neighbor and furthest neighbor in HCA were performedbased on nine investigated components from30tested samples. Consistentresults were obtained to show that30tested A. asphodeloides samples couldbe successfully grouped into two main clusters or domains (I and II) by bothmethods. HB-4, HB-5, and HB-6were in cluster (domain) II and the othersamples were in cluster (domain) I, which was further divided into twosubgroups (A and B).Conclusion: The validation results of the method indicated that themethod was simple, rapid, specific, and reliable. The results demonstrated thatthe quantitative difference in content of nine active compounds was useful notonly for chemotaxonomy of many samples from different sources but also forthe standardization and differentiation of many similar samples. Simultaneousquantification of bioactive components by HPLC-ESI-MS coupled withchemometric techniques would be a well-acceptable strategy tocomprehensively control the quality of A. asphodeloides.Part two A comparative study on the pharmacokinetics of a traditionalChinese herbal preparation with the single herb extracts inrats by LC–MS/MS methodObjective: The Er-Mu preparation (EMP) is a well-known traditionalChinese prescription that has been clinically employed for the treatment ofasthma and bronchial inflammation for hundreds of years. It is prepared fromZhimu (Anemarrhenae rhizome, ARR) and Chuanbeimu (Fritillariaecirrhosae bulbus, FCB) at the weight ratio of1:1. Neomangiferin, mangiferin,peimine, peiminine, timosaponin BII and timosaponin AⅢ are the majoractive ingredients of EMP for their anti-inflammatory or anti-asthmatic effects.The aim of this study was to investigate the pharmacokinetics of the targetcompounds from the recipe of EMP and the single herb extracts ofAnemarrhenae asphodeloides Bge.(ARR) and Fritillariae cirrhosae D.Don(FCB), and the influence of compatibility on the pharmacokinetics of the mainactive ingredients. Methods: ARR (100g) and FCB (100g) were extracted three times byrefluxing with70%ethanol (1:10,1:10and1:5, w/v) for1h per time,respectively. The extraction solutions were combined to be filtered and thenethanol was removed under reduced pressure. Finally, the residuary solutionwas condensed to2g/mL crude drug. ARR and FCB extracts were mixed atthe ratio of1:1to constitute the prescription of EMP. To calculate theadministered dose, the contents of six components in the decoctions werequantitatively determined by the external standard method with the samechromatography conditions as described in section “Liquid chromatography”and “Mass spectrometer”. The contents ofneomangiferin, mangiferin,timosaponin BII and timosaponin A in the ARR extract were8.76,21.16,74.24and4.16mg/mL, respectively. The contents of peimine and peimininein the FCB extract were0.32and0.57mg/mL, respectively.The rats were randomly assigned to three groups and orally administeredwith the single herb extracts of ARR and FCB, and the recipe of EMPrespectively. Blood samples were collected from the fossa orbitalis vein intoheparinized centrifuge tubes at5,10,15,30,60,120,180,240,360,480,600,1440,2160and2880min after the single oral administration of threeextracts.The concentrations of the target compounds in rat plasma weredetermined by an optimal liquid chromatography–electrospray ionization massspectrometry (HPLC-ESI-MS）and multiple reaction monitoring (MRM) witha multi-switching monitoring mode coupled with simple protein precipitationmethod, and the main pharmacokinetic parameters were estimated. A lineargradient elution of eluents A (acetonitrile) and B (water containing0.1%formic acid) was used for separation. The elution programmer was optimizedand conducted as follows: a linear gradient of5-55%A with the range of0.0-7.0min, a linear gradient of55-95%A with the range of7.0-10.0min,then holding this mobile phase ratio for5min, a linear gradient of95-5%Awith the range of15.0-15.2min. This was followed by6min equilibrationperiod prior to the injection of each sample. The solvent flow rate was kept at0.8mL/min. The determination was divided into3periods. With the range of 0.0-6.19min, neomangiferin and mangiferin were detected in negativeionization mode, then the negative ionization mode was switched to positivemode in the range of6.19-6.22min. After that, timosaponin BII, timosaponinA, peimine, peiminine and IS were detected in positive mode in the range of6.22-15.0min. All the data were processed by non-compartmental analysiswith Excel software. The pharmacokinetic parameters, such as maximumplasma concentration (Cmax) and time of maximum concentration (Tmax),were directly obtained from the plasma concentration-time plots. Theelimination rate constants (k) were determined by the linear regressionanalysis of the logarithmic transformation of the last four data points of thecurve. The elimination half-life (T1/2) was calculated with the followingequation: T1/2=0.693/k. Statistical significance was assessed by an unpairedStudent’s t-test and the significance level of P<0.05was adopted for allstatistical comparisons. All results were expressed as arithmeticmean±standard deviation (S.D)Results: The developed HPLC-ESI-MS method by switching positiveand negative ESI sources in a single run was successfully applied to study thepharmacokinetics of six compounds in SD rat, which was powerful in terms ofsensitivity, selectivity, time savings and solvent consumption in quantitativeanalysis of complex herbal medicines. The LLOQ for neomangiferin,mangiferin, peimine, peiminine, timosaponin BII and timosaponin A were4.63,2.92,3.56,4.44,4.40and4.00ng/mL, which was sensitive enough forthe pharmacokinetic study of the analytes in rats. The precisions (RSD) of theanalytes were all less than7.4%and7.8%. The dilution integrity precision ofthe QC samples was less than8.9%and the accuracy was in the range from-6.8%to12.4%. The results of stability offered satisfactory stability with theaccuracy in the range from-9.6%to9.9%. The mean extraction recoveries ofthe investigated components in plasma at three different concentration levelswere found to be80.6~102.3%with RSD less than5.7%. The matrix effectvalues obtained for analytes ranged from87.2to106.8%, and the matrix effecton IS was97.8%. Significant differences (p <0.05) was found in the pharmacokineticparameters of neomangiferin, mangiferin, peimine and peiminine betweensingle ARR or FCB extract and the combination treatment (P<0.05).WhenEMP was administered orally to rats, Cmax and AUC of mangiferin andneomangiferin were increased significantly, and T1/2was slightly shorterwithout affecting Tmax noticeably by comparison to the administration ofARR extract alone. There was no statistically significant difference in thepharmacokinetic parameters of timosaponin BII and timosaponin A betweenthe single ARR extract group and the combination group. When EMP wasorally administered to rats, Cmax and AUC of peimine and peiminine wereincreased significantly, and T1/2was obviously prolonged without affectingTmax noticeably by comparison to the administration of single FCB extract.Conclusion: The simple and rapid method was successfully applied tothe pharmacokinetic study in rats after the intragastric administration of EMPand single herb extracts, which would provide a methodical application formore extracts and preparations. It can be concluded from the results of thisstudy, there was significant difference in the pharmacokinetic parameters ofxanthone glycosides and isosteroidal alkaloids after the oral administration ofsingle extracts and EMP. This study indicated that formula compatibility couldsignificantly affect the pharmacokinetics of some components in rat plasma.Our study has preliminarily elucidated of the priority in compatibleadministration of EMP based on pharmacokinetic studies, which also providesa basis for formula compatibility studies and further clinical pharmacokineticsevaluation of TCM preparation. It was surmised that formula compatibilitycould significantly influence the pharmacokinetics of EMP and our study haspreliminarily elucidated the priority in the compatible administration of EMPbased on pharmacokinetic studies.Part three Simultaneous quantification of six bioactive constituents inrat bile and urine after oral administration of a traditionalChinese herbal preparation by HPLC-MS/MS method andits application to excretion studyObjective: The Er-Mu preparation (EMP) is a well-known traditionalChinese prescription that has been clinically employed for the treatment of asthma and bronchial inflammation for hundreds of years. It is prepared fromZhimu (Anemarrhenae rhizome, ARR) and Chuanbeimu (Fritillariaecirrhosae bulbus, FCB) at the weight ratio of1:1. Neomangiferin, mangiferin,peimine, peiminine, timosaponin BII and timosaponin AⅢ are the majoractive ingredients of EMP for their anti-inflammatory or anti-asthmatic effects.A sensitive and selective HPLC–ESI–MS/MS method for simultaneousdetermination of six major compound (neomangiferin, mangiferin,timosaponin AⅢ, peimine and peiminine) in rat bile and urine. Using thismethod, the biliary and urine excretion profiles of these compounds werefurther investigated after a single oral administration of single ARR, FCBextracts and EMP.Methods: ARR (100g) and FCB (100g) were extracted three times byrefluxing with70%ethanol (1:10,1:10and1:5, w/v) for1h per time,respectively. The extraction solutions were combined to be filtered and thenethanol was removed under reduced pressure. Finally, the residuary solutionwas condensed to2g/mL crude drug. ARR and FCB extracts were mixed atthe ratio of1:1to constitute the prescription of EMP. To calculate theadministered dose, the contents of six components in the decoctions werequantitatively determined by the external standard method with the samechromatography conditions as described in section “Liquid chromatography”and “Mass spectrometer”. The contents ofneomangiferin, mangiferin,timosaponin BII and timosaponin A in the ARR extract were8.76,21.16,74.24and4.16mg/mL, respectively. The contents of peimine and peimininein the FCB extract were0.32and0.57mg/mL, respectively. After theprepared three extracts were orally administered to three groups (six in eachgroup) at a dose of17.52,42.32,0.64,1.14,148.48and8.32mg/kg ofneomangiferin, mangiferin, peimine, peiminine, timosaponin BII andtimosaponin AⅢ respectively, all the rats were placed in metabolic cages.After oral administered, urine samples were collected during0–4,4–8,8–12, 12–24,24–36,36–48,48–60and60–72h periods. After the prepared threeextracts were orally administered to three groups (six in each group) at a doseof17.52,42.32,0.64,1.14,148.48and8.32mg/kg of neomangiferin,mangiferin, peimine, peiminine, timosaponin BII and timosaponin AⅢrespectively, all the rats were anesthetized using ethyl carbamate and weresurgically implanted with a cannula in the bile duct. Bile samples werecollected at2,4,6,8,10,24,36and48h. The blank bile and urine werecollected before dosing. The volumes of urine and bile samples were recorded.The urine and bile samples were centrifuged at3,000rpm for10min, and thenall supernatants were kept at20°C until use.A simple direct injection method was applied to extract the sixcompouds and IS from rat bile and urine. The chromatographic separation wasperformed on a Diamonsil C18column (250mm×4.6mm,5m), and thecolumn temperature was operated at room temperature. A linear gradientelution of acetonitrile and water was used for the separation. The analyseswere performed using an electrospray ionization source in positive andnegative mode respectively. Multiple–reaction monitoring (MRM) mode wascarried out for obtaining the maximum sensitivity for the detection of thetarget compounds. A linear gradient elution of eluents A (acetonitrile) and B(water containing0.1%formic acid) was used for separation. The elutionprogrammer was optimized and conducted as follows: a linear gradient of5-55%A with the range of0.0-7.0min, a linear gradient of55-95%A with therange of7.0-10.0min, then holding this mobile phase ratio for5min, a lineargradient of95-5%A with the range of15.0-15.2min. This was followed by6min equilibration period prior to the injection of each sample. The solventflow rate was kept at0.8mL/min. The determination was divided into3periods. With the range of0.0-6.19min, neomangiferin and mangiferin weredetected in negative ionization mode, then the negative ionization mode wasswitched to positive mode in the range of6.19-6.22min. After that,timosaponin BII, timosaponin AⅢ, peimine, peiminine and IS were detectedin positive mode in the range of6.22-15.0min. Results: A rapid HPLC-ESI-MS/MS method was established for thesimultaneous quantification concentrations of six analytes in bile and urine.Characteristics of cumulative urinary and biliary excretion of six analyteswere determined. The data of cumulative excretion amount and recoverycumulative excretion of the six analytes in urine and bile were shown in table6. The recovery cumulative excretion of neomangiferin, mangiferin, peimine,peiminine, timosaponin B Ⅱ and timosaponin AⅢ in the urine and bile afteroral administration of single extracts or EMP were presented in Fig.6. Theresults showed that less than7%analytes were excreted as unchanged drug viaurine and less than5%analytes was excreted as prototype via bile, suggestingthat these six analytes undergo extensive metabolism in the body eitheradministrated with EMP or with the single extracts. For the comprehensiveevaluation of the biotransformation of six analytes, quantitation of thesebioactive ingredients in vivo is crucial. Therefore, the qualitative analysis isunder further study.It could be concluded that the six analytes showed significantly differentexcretion behaviors between EMP and the single extracts. The urinaryexcretion level of the six analytes was significantly higher (approximatelyfourfold to forteenfold) in EMP than that in single extracts. In the bile theaverage percentages of mangiferin, peimine, peiminine excreted over the doseadministered with EMP were higher than that with the single extracts, whilethe others after administered with EMP were less than that with ARR or FCB.Different metabolic mechanism between EMP and single extracts mightcontribute to the different results, which need further study.Conclusion: The specificity, linearity, accuracy, precision, recovery,matrix effect and several of stabilities have been validated for the six analytesin rat bile and urine samples. The results showed that this method is robust,specific and sensitive and it can successfully fulfill the requirement ofexcretion study of the six analytes in EMP and the single extracts. In this study,a sensitive and selective LC-MS/MS method has been developed andvalidated for the simultaneous determination of six analytes in rat urine and bile. The method was used to describe urinary and biliary excretion–timeprofiles and cumulative excretion of the analytes after oral administration ofthe three extracts. To our knowledge, this is the first comparative excretionstudy after oral administration of EMP and its single extracts. These resultsmight be helpful for further in vivo study and clinical application of EMP.Part four Simultaneous Determination of Seven components inJinlianqingre Granule by HPLC-MSObjective: To develop a method for the determination of orientin (1),vitexin (2), hyperin (3), timosaponin BII (4), timosaponin A Ⅲ(5), mangiferin(6) and neomangiferin (7) in Jinlianqingre granule by HPLC-MS.Methods: The samples were separated on a Diamonsil C18column (4.6mm×150mm,5μm) by gradient elution using acetonitrile and0.1%aqueousformic acid as the mobile phase at a flow rate of800μL/min. The columntemperature was30℃. Multiple-reaction monitoring (MRM) scanning wasemployed for quantification with with switching electrospray ion sourcepolarity between positive and negative modes in a single run. The elutionprogram was optimized as follows:0–2.5min, linear change from A–B(5:95,v/v) to A–B (55:45, v/v);2.5–5min, linear change from A–B (55:45, v/v) toA–B (95:5, v/v); and5–15min, isocratic elution A–B (95:5, v/v); then quicklyreturned to initial A–B (5:95, v/v). This was followed by the equilibrationperiod of6min prior to the injection of each sample. The flow rate of mobilephase was set at0.8mL/min and the injection volume was10μL.Results: The complete separation was obtained within15min for theseven compounds. The regression equations showed linear relationshipsbetween the peak area and content of each compound. The average recoveriesof the compounds listed above were99.34%,99.28%,101.6%,101.3%,100.5%,101.9%and98.86%(n=3), and the RSDs were1.12%,1.36%,1.57%,1.74%,1.52%,1.43%and0.92%, respectively.Conclusion: The method is simple, accurate and highly reproducible, andcan be used for the determination of seven compounds in Jinlianqingregranule. Part five Simultaneous Determination of nine components inZhibaidihuang Concentrated pill by HPLC-MSObjective: To develop a method for the determination ofneomangiferin(1), mangiferin (2), timosaponin BII (3), timosaponin A Ⅲ(4),berberine (5), morroniside (6), loganin (7), paeoniflorin (8) and paeonol (9)inZhibaidihuang concentrated pill by HPLC-MS.Methods: The samples were separated on a Diamonsil C18column (4.6mm×150mm,5μm) by gradient elution using acetonitrile and0.1%aqueousformic acid as the mobile phase. The column temperature was30℃. Theelution program was optimized as follows:0–2.5min, linear change fromA–B(5:95, v/v) to A–B (55:45, v/v);2.5–5min, linear change from A–B(55:45, v/v) to A–B (95:5, v/v); and5–15min, isocratic elution A–B (95:5,v/v); then quickly returned to initial A–B (5:95, v/v). This was followed by theequilibration period of6min prior to the injection of each sample. The flowrate of mobile phase was set at0.8mL/min and the injection volume was10μL. Multiple-reaction monitoring (MRM) scanning was employed forquantification with with switching electrospray ion source polarity betweenpositive and negative modes in a single run.Results: There was significant correlation between the ratio of peak areaand the concentration of each compound within the test ranges. The averagerecoveries (n=3) of the nine compounds listed above were97.4%,99.2%,100.3%,101.8%,99.5%,101.6%,99.6%,101.7%and98.86%. The RSDswere1.2%，1.6%，1.0%，0.7%，1.5%，0.4%，1.0%，1.1%and0.9%,respectively.Conclusion: The method is simple, accurate and highly reproducible, andcan be used for the determination of nine compounds in ZhibaidihuangConcentrated pill. |
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