Multicomponent Analysis And Pharmacokinetic Studies By UPLC/Q-TOF-MS/MS In Qiliqiangxin Capsules

Qiliqiang capsules (QL) is a newly developed Chinese patent drugaccording to collateral disease theory, which was widely used in the treatmentof chronic heart failure. QL has been used over a decade in clinical because itwas China’s first patent drug with the support of numerous research evidencesin evidence-based medicine field and the exact effects in treatment of chronicheart failure. QL is a specific TCM extract obtained from11types of herbs,including astragali radix, ginseng radix et rhizoma, aconiti lateralis radixpreparata Salvia miltiorrhiza radix et rhizoma, semen descurainiae lepidii,alismatis rhizoma, polygonati odorati rhizoma,cinnamomi ramulus, carthamiflos, periploca cortex, and citri reticulatae pericarpium. Astragali radix andaconiti lateralis radix preparata are the principal pharmacologically activecomponents. Saponins, flavonoids, phenolic acids, alkaloids, etc are the mainchemical compotents. Until now, most studies of QL capsule have focused onits clinical efficacy and pharmacology, in contrast, its chemical compositionanalysis and quality control were rarely concerned, and there was no researchfocusing on its toxic effective composition aconitum alkaloids.In thispaper,we developed a fast and efficient, high sensitivity, strong specificityUPLC/Q-TOF-MS/MS as the mainly analysis method to study thecharacteristics of composition in QL, blood transitional components, andquantitatively analysis of nine main efficacy aconitum alkaloids componentsin vivo and pharmacokinetic study in rats, in order to provide a reference forfurther clarify efficacy material base.Part1Component analysis of QL by UPLC/Q-TOF-MS/MSObjective: To establish an ultra high performance liquid chromatographycoupled with tandem quadrupole time-of-flight mass spectrometer detection(UPLC/Q-TOF-MS/MS) comprehensive quickly clarify QL of chemical composition analysis method.Methods: For sample preparation,0.3g of the QL intermediates and0.3g dry Fuzi powder were precisely weighed and placed in a stoppered brownvolumetric flask. Then10mL50%methanol was added, the flask wasweighed, and the mixture was filtered for30min and ultrasonically extractedfor30min (power,250W; frequency,40kHz). After the mixture was cooledand weighed,50%methanol was added to compensate for the weight loss, andthis mixture was centrifuged at15300rpm for10min; the supernatant wasfiltered through a0.22μm membrane and used foranalysis.UPLC/Q-TOF-MS/MS analysis was performed using the Agilent1290UPLC system (Agilent Technologies, USA) coupled with the AB SCIEXTripleTOFTM5600+MS system (AB SCIEX, USA) integrating a switchableelectrospray ion source interface. A Phenomenex Kinelex C18(2.1mm×100mm,2.7μm) reversed-phase column equipped with a Phenomenexultra-efficient C18guard column was used for chromatography. The mobilephase consisted of0.1%formic acid in water (A) and acetonitrile (B), andgradient elution was performed via the following steps:(0～0.5) min,10%B;(0.5～2.5) min,10%→40%B;(2.5～9) min,40%→70%B;(9～14) min,70%→90%B;(14～15) min,90%→100%B;(15～17) min,100%B;(17～17.1) min,100%→10%B;(17.1～19) min,10%B. The columntemperature was40°C; flow rate,0.4–400μL/min; and injection volume,5μL.The liquid phase conditions for separation of the9alkaloids from QL andaconite extraction were the same as those mentioned above. The conditions ofMS/MS detector were as follows: ion spray voltage,5.5kV/-4.5kV; ion sourcetemperature,550°C; declustering potential(DP),60V/-55V; collisionenergy(CE),45eV/-40eV. Nitrogen was used as the nebulizer and theauxiliary gas, and the nebulizer gas (gas1), the heater gas (gas2) and thecurtain gas were set to55,55and35psi. The complete scan was performed inthe ESI-positive ion mode and ESI-negative within the m/z100–1500amumass range and in a cumulative time of250ms. For theinformation-dependent acquisition standards of each analyte, the8strongest fragment ions over100cps underwent ion scan within the abovementionedmass range, at a cumulative time of70ms. The collision voltage differencewas15eV/20eV, and dynamic background subtraction was open. Anautomatic calibration system (CDS) was then used for automatic tuning andcalibration of the MS and MS/MS. Data acquisition and processing analysiswere conducted using the Analyst TF1.6software, PeakView1.2software,and MultiQuant2.1.1software(AB Sciex). The full-scan UPLC/Q-TOF-MS/MS chromatogram and the chromatogram produced using in-sourcecollision-induced dissociation were acquired，respectively．With reference toreference substance chromatography and mass spectrometry information andthrough calculating accuracy mass and isotopicfit value of the molecular ioncluster，molecular formula was confirmed，and further the matched compoundwas researched in the established database composed of the knownconstituents in QL or11single herbs come from it’s compoundprescription．Finally，the peaks were identified by elucidating the massspectrum produced using in-source collision-induced dissociation．Results: A rapid and efficient HPLC-TOF/MS method were developedto determine the chemical constituents in QL.,and139chromatographic peakswere identified in QL chromatograms, main ingredients including triterpenoidsaponins, flavonoids, alkaloids, phenolic acids, terpenoids, etc. This studycompared comprehensively expounds the chemical composition of QL, forQL efficacy material base research and laid a solid foundation for qualitycontrolConclusion: This method is concluded rapid，sensitive and accuracy，andit can be used for analysis of the constituents in Qliqiangxin capsules and11single herbs come from it’s compound prescription． This study also providesa provides a train of thought to the components analysis of other traditionalChinese medicines．Part2Study on Fingerprint of Medicated Serum of Qiliqiangxin capsuleObjective：To establish Qiliqiangxin capsule（QL）medicated serumfingerprint，in order to distinguish the drug-induced composition and metabolism product from Serum of rats administered QL，basising on serummedicine chemistry and efficacy material foundation.Methods: The medicine serum was prepared after the rats were oralyadministrated QL. Serum of rats administered QL fingerprint was establishedby UPLC and compared with the fingerprints of blank serum，medicine invitro and every single herb medicine，and the ownership of every peak wasanalyzed.Results:13compositions were detected in QL medicated serumfingerprint，4were drug-induced composition.Conclusion: This method is accurate，reliable and reproducible，whichcan be used to research serummedicine chemistry and efficacy materialfoundation.Part3Study on adscription of plasma effective constituents of rats afteradministrated with Qiliqiangxin capsules by UPLC/Q-TOF-MS/MSObjective: To establish an ultra high performance liquid chromatographycoupled with tandem quadrupole time-of-flight mass spectrometer detection(UPLC/Q-TOF-MS/MS) comprehensive quickly analysis method to clarify theMethods: The medicine serum was prepared after the rats were oralyadministrated QL. The effective constituents of Serum of rats administered QLwas anlysized by UPLC/Q-TOF-MS/MS,compared with the blank serum，medicine in vitro and every single herb medicine，and the ownership of everypeak was analyzed. The information on the total ion chromatogram，masschromatogram and the mass spectrogram were synthetically analyzed toconfirm the effective constituents absorbed into blood.Results:61constituents of QL were detected in the rats plasma post theintragastric administration of QL，among which34were original consitunentscame from QL，and the others might be metabolits of original consitutents.Conclusion: The findings abtained from the study can provide the usefulinformation for the determination of bioactive substances of the QL. Part4Determination of9Aconitum Alkaloids in Qiliqiangxin Capsuleand its Principal Drug Fuzi Using UPLC/Q-TOF-MS/MS AnalysisObjective: To develop a special ultra-performance liquidchromatography/quadrupole time of flight mass spectrometry/massspectrometry method for rapid quantitative analysis of9aconitum alkaloids inQL and its principal drug Fuzi,namely, fuziline, neoline, talatisamine,aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine,and benzoylmesaconineMethods: UPLC/Q-TOF-MS/MS analysis was performed using theAgilent1290UPLC system (Agilent Technologies, USA) coupled with the ABSCIEX TripleTOFTM5600+MS system (AB SCIEX, USA) integrating aswitchable electrospray ion source interface. A Phenomenex Kinelex C18(2.1mm×100mm,2.7μm) reversed-phase column equipped with a Phenomenexultra-efficient C18guard column was used for chromatography. The mobilephase consisted of0.1%formic acid in water (A) and acetonitrile (B), andgradient elution was performed via the following steps:0–1min (10%B),1–2min (10–30%B),2–6min (30–40%B),6–7min (40–100%B),7–8min(100%B),8–8.1min (100–10%B),8.1–10min (10%B). The columntemperature was40°C; flow rate,0.4–400μL/min; and injection volume,5μL.The conditions of MS/MS detector were as follows: ion spray voltage,5.5kV;ion source temperature,550°C; declustering potential (DP),60V; collisionenergy (CE),35eV. Nitrogen was used as the nebulizer and the auxiliary gas,and the nebulizer gas (gas1), the heater gas (gas2) and the curtain gas wereset to50,50and35psi. The complete scan was performed in the ESI-positiveion mode within the m/z100–1500amu mass range and in a cumulative timeof250ms. For the information-dependent acquisition standards of eachanalyte, the8strongest fragment ions over100cps underwent ion scan withinthe abovementioned mass range, at a cumulative time of70ms. The collisionvoltage difference was20eV, and dynamic background subtraction was open.An automatic calibration system (CDS) was then used for automatic tuningand calibration of the MS and MS/MS. Data acquisition and processing analysis were conducted using the AnalystTF1.6software, PeakView1.2software, and MultiQuant2.1.1software(AB Sciex).Results: All analytes, namely, fuziline, neoline, talatisamine, aconitine,hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, andbenzoylmesaconine, were examined simultaneouslyiwithin10min withoutthe need for baseline separation. All analytes had good linearity within thedetection range (r2>0.9940), the analysis was repeatable (RSD <4.34%), theinter-and intra-day precision were good (RSD <4.83%), and the recovery rateranged from94.75%to106.21%. Our novel method for the examination of the9aconitum alkaloids in QL and Fuzi was found to be simple, accurate, reliable,and rapid.Conclusion: The validation results of the method indicated that themethod was simple, rapid, specific, and reliable. In the present study, themajor compounds in QL were quantitatively analyzed for the first time. Theresults demonstrated that QL compound compatibility can change thecomposition ratio and total alkaloid content in aconite alkaloids so as to obtainthe attenuated efficiency of aconite alkaloids. The novelUPLC/Q-TOF-MS/MS method developed here allowed fast, simple, andreliable simultaneous quantitative detection of the9major chemicalcompounds in QL capsules (fuziline, neoline, talatisamine, aconitine,hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, andbenzoylmesaconine). This quantitative method has the advantages of highseparation, high sensitivity, high selectivity, and a short analysis time, makingit suitable even for evaluation of the quality of traditional Chinese herbalcompounds.Part5A study on the pharmacokinetics of9Aconitum Alkaloids inQiliqiangxin capsule in rats by UPLC/Q-TOF-MS/MS methodObjective: The aim of this study was to establish a UPLC/Q-TOF-MS/MS method to investigate the pharmacokinetics of the target compoundsnamly fuziline, neoline, talatisamine, aconitine, hypaconitine, mesaconitine,benzoylaconine, benzoylhypaconine, and benzoylmesaconine from Qiliqiangxin capsules in rats in vivo, and to establish their medicine-curve,obtained pharmacokinetic parameters and characteristics, provide referencefor clinical medication.Methods: After fasting12hour，the SD rats were orally administratedqiliqiangxin,and were blooded respectively before administration and0.25,0.5,1,2,4,6,8,10,12,24,36,48h after administeration orally by anesthesiaabdominal aorta centrifuge tube by heparin,3000r/min, the centrifugal10minutes, the separation of plasma, using solid phase extraction pretreatment ofsamples,with DXP as internal standard.UPLC/Q-TOF-MS/MS analysis wasperformed using the Agilent1290UPLC system (Agilent Technologies, USA)coupled with the AB SCIEX TripleTOFTM5600+MS system (AB SCIEX,USA) integrating a switchable electrospray ion source interface. APhenomenex Kinelex C18(2.1mm×100mm,2.7μm) reversed-phasecolumn equipped with a Phenomenex ultra-efficient C18guard column wasused for chromatography. The mobile phase consisted of0.1%formic acid inwater (A) and acetonitrile (B), and gradient elution was performed via thefollowing steps:0-1min，5%B；1~3min，5%→10%B；3~13min，10%→60%B；13~13.5min，60%→5%B；13.5~15min，5%B。The columntemperature was40°C; flow rate,0.4–400μL/min; and injection volume,5μL.The conditions of MS/MS detector were as follows: ion spray voltage,5.5kV;ion source temperature,550°C; declustering potential(DP),60V; collisionenergy(CE),44eV. Nitrogen was used as the nebulizer and the auxiliary gas,and the nebulizer gas (gas1), the heater gas (gas2) and the curtain gas wereset to50,50and35psi. The complete scan was performed in the ESI-positiveion mode within the m/z50–1500amu mass range and in a cumulative time of250ms. For the information-dependent acquisition standards of each analyte,the8strongest fragment ions over100cps underwent ion scan within theabovementioned mass range, at a cumulative time of70ms. The collisionvoltage difference was15eV, and dynamic background subtraction was open.An automatic calibration system (CDS) was then used for automatic tuningand calibration of the MS and MS/MS. Data acquisition and processing analysis were conducted using the Analyst TF1.6software, PeakView1.2software, and MultiQuant2.1.1software(AB Sciex). All the data wereprocessed by non-compartmental analysis with Excel software. Thepharmacokinetic parameters, such as maximum plasma concentration (Cmax)and time of maximum concentration (Tmax), were directly obtained from theplasma concentration-time plots. The elimination rate constants (k) weredetermined by the linear regression analysis of the logarithmic transformationof the last four data points of the curve. The elimination half-life (T1/2) wascalculated with the following equation: T1/2=0.693/k. All results wereexpressed as arithmetic mean±standard deviation.Results: All analytes, namely, fuziline, neoline, talatisamine, aconitine,hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, andbenzoylmesaconine, were examined simultaneously within15min without theneed for baseline separation. All analytes had good linearity within thedetection range (r2>0.9946), the analysis was repeatable (RSD <4.34%), theinter-and intra-day precision were good (RSD=0.4%~5.0%), and the averagerecovery rate ranged from85.70%to97.37%, The matrix effect valuesobtained for analytes ranged from94.63to99.32%, and the matrix effect on ISwas93.95%. The LLOQ for fuziline, neoline, talatisamine, aconitine,hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, andbenzoylmesaconine was0.05,0.063,0.152,0.160,0.205,0.247,0.053,0.240and0.154ng/mL, which sensitive enough for the pharmacokinetic study of theanalytes in rats. The results of stability offered satisfactory stability with theaccuracy in the range from1.06%to4.26%. When qiliqiangxin capsule wasadministered orally to rats, fuziline, neoline, talatisamine, aconitine,hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, andbenzoylmesaconine, were absorbed and spread out quickly in the body. andbimodal appeared in medicine curve, whih was consistent with literaturereports. Compared to eliminate flat, we speculate that metabolic characteristicswith alkaloids itself, compound compatibility and too high dose related. Conclusion: This method is simple and rapid,and it was successfullyapplied to the pharmacokinetic study of9analytes, namly fuziline, neoline,talatisamine, aconitine, hypaconitine, mesaconitine, benzoylaconine,benzoylhypaconine, and benzoylmesaconine, after the intragastricadministration of QL in rats. It can be concluded from the pharmacokineticresults of this study, qiliqiangxin capsule compound compatibility may affectthe pharmacokinetic characteristics of active ingredients, and couldpreliminary inference the rationality of the compound compatibility.