基于液质联用技术的芪苈强心胶囊中多组分分析与药代动力学研究
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摘要
芪苈强心胶囊是中医络病理论指导研发的临床广泛用于慢性心力衰竭的中药新药，临床使用已超过十年，为中国首个具有循证医学研究证据、疗效确切的治疗慢性心衰的中成药。芪苈强心胶囊由黄芪、附子、人参、丹参、葶苈子、泽泻、红花、玉竹、陈皮、桂枝、香加皮等十一味中草药提取而成，其化学成分主要有皂苷类、黄酮类、酚酸类、生物碱等。目前关于芪苈强心胶囊研究多集中在临床疗效和药理学研究方面，化学成分分析和质量控制研究很少，也未有药物代谢动力学研究的报道。本文采用UPLC/Q-TOF-MS/MS作为主要分析方法，利用其快速高效、灵敏度高、特异性强的特点，对芪苈强心胶囊化学成分、血中移行成分进行定性鉴别，并对其9个主要药效成分即乌头生物碱进行定量分析与大鼠体内的药代动力学研究，为进一步阐明药效物质基础提供理论依据和参考价值。
第一部分UPLC/Q-TOF-MS/MS法鉴定芪苈强心胶囊中化学成分
目的：建立一种超高效液相色谱与串联四极杆飞行时间质谱仪联用技术(UPLC/Q-TOF-MS/MS)全面快速地阐明芪苈强心胶囊的化学组成的分析方法。
方法：取芪苈强心胶囊样品研细，取约0.3g，精密称定，置具塞锥形瓶中，加入50%甲醇10mL，超声处理30min，放冷，滤过，用50%甲醇补足至原重量后，摇匀，16000r/min离心10min，取上清夜，过滤（0.22μm），取滤液进样分析。色谱条件：色谱柱：Phenomenex Kinetex C18100×2.1mm,2.6μm。流动相为0.1%甲酸水溶液(A)-乙腈(B)，梯度洗脱程序为：(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。柱温：40℃；流速：400μL/min；进样体积：5μL。质谱色谱条件：离子化模式为电喷雾正、负离子模式，正负离子源电压分别为5500V/-4500V，离子源温度为550℃，裂解电压（DP）分别为60V/-55V，碰撞能量（CE）分别为45eV/-40eV，碰撞能量扩展（CES）分别为15eV/20eV。雾化气体为氮气，辅助气1为55PSI，辅助气2为55PSI，气帘气为35PSI。一级质谱母离子扫描范围为100-1500，IDA设置响应值超过100cps的8个最高峰进行二级质谱扫描，子离子扫描范围为50-1500，开启动态背景扣除（DBS）。数据采集软件：Analyst TF1.6software（AB SCEIX，USA）；数据处理软件系统：Peakview1.2software（AB SCEIX，USA）。获取UPLC/Q-TOF MS/MS全扫描质量色谱图和源内裂解的UPLC-TOF/MS色谱图，参照对照品信息或通过分子离子精确质量和同位素拟合度的分析确定分子式，在数据库中检索匹配的化合物，进而解析源内裂解的质谱图，从而对各色谱峰进行指认。并在相同实验条件下对芪苈强心胶囊及其组方各单味药的色谱图进行比对分析,归属色谱峰来源。
结果：从芪苈强心胶囊色谱图中共鉴定了139个色谱峰，主要成分包括三萜皂苷类、黄酮类、生物碱类、酚酸类、萜类等。本研究比较全面地阐明了芪苈强心胶囊的化学组成，为芪苈强心胶囊的药效物质基础研究和质量控制奠定了基础。
结论：该方法简便、快速，灵敏度，可用于芪苈强心胶囊血中移行成分定性分析。
第二部分芪苈强心胶囊UPLC血清指纹图谱研究
目的：采用超高效液相色谱(UPLC)法建立芪苈强心胶囊血清指纹图谱，为芪苈强心胶囊血清药物化学及药效物质基础研究提供依据。
方法：大鼠灌胃给药制备含药血清，采用Waters ACQUITY UPLCBEH C18色谱柱(2.1mm×100mm，1.7μm)，流动相乙腈-0.1%甲酸水，梯度洗脱，流速0.5mL·min-1，柱温40℃，检测波长280nm，分析芪苈强心胶囊大鼠含药血清，并与相同条件下空白血清、体外全方及其11个单味药图谱比对，对色谱峰归属进行分析。
结果：建立了芪苈强心胶囊的UPLC血清指纹图谱，共标定了13个共有峰，指认了其中4个共有峰，均为药源性成分，来自香加皮。10批芪苈强心胶囊血清指纹图谱的相似度均在0.868～1.000。
结论：本研究建立的方法准确、可靠、重现性好，可用于芪苈强心胶囊血清药物化学及药效物质基础的研究。
第三部分UPLC/Q-TOF-MS/MS法鉴定芪苈强心胶囊血中移行成分
目的：建立一种超高效液相色谱与串联四极杆飞行时间质谱仪联用技术(UPLC/Q-TOF-MS/MS)全面快速地阐明芪苈强心胶囊血中移行成分的分析方法。
方法：采用UPLC/Q-TOF-MS/MS方法，对比芪苈强心胶囊、空白血清、芪苈强心胶囊含药血清总离子流图，质谱图等信息，通过对比各色谱峰的保留时间及质谱图裂解规律，确认大鼠灌胃芪苈强心胶囊内容物后血中移行成分及其归属。
结果：大鼠口服芪苈强心胶囊后血中共发现61个入血成分，其中34个为芪苈强心胶囊中原形成分，27个为原形药物的代谢产物。本研究将为阐明芪苈强心胶囊药效物质基础提供依据。
结论：该方法简便、快速，灵敏度，可以用于芪苈强心胶囊化学成分定性分析。
第四部分UPLC/Q-TOF-MS/MS法快速测定芪苈强心胶囊及其附子中的9个乌头类生物碱成分含量
目的：建立一种简便、准确、可靠的UPLC/Q-TOF-MS/MS分析方法，用于芪苈强心胶囊和附子中9个乌头类生物碱成分的含量测定。
方法：取芪苈强心胶囊样品研细，取约0.3g，精密称定，置具塞锥形瓶中，加入50%甲醇10mL，超声处理30min，放冷，滤过，用50%甲醇补足至原重量后，摇匀，16000r/min离心10min，取上清夜，过滤（0.22μm），取滤液进样分析。色谱条件：色谱柱：Phenomenex Kinetex C18100×2.1mm,2.7μm。流动相由0.1%甲酸水（A）和乙腈（B）组成，梯度洗脱：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).柱温：40℃；流速：400μL/min；进样体积：5μL。每次进样预平衡5min，再进行梯度洗脱。质谱色谱条件：离子喷雾电压，5.5KV；离子源温度,550℃;解簇电压(DP)，60V和碰撞能量(CE)，35eV。氮气为雾化气和辅助气，雾化气50psi，辅助气50psi，气帘气35psi。在m/z100-1500amu质量范围ESI正离子模式下进行全扫描，积累时间250ms。IDA采用标准：每个分析物，超过100cps的八个最强的碎片离子在100~1500amu质量范围内进行子离子扫描，累积时间70ms。碰撞电压差20eV，动态背景扣DBS开启。自动校准系统(CDS)对MS和MS/MS自动进行调谐和校正。数据获取和处理分析采用Analyst TF1.6软件、PeakView1.2软件和MultiQuant2.1.1软件。
结果：在一定浓度区间内9种分析物的线性关系良好，r2>0.9940；所有分析物定量限LOQ范围0.03~0.50ng/mL，日内精密度、日间精密度RSD均小于3.38%，重复性试验RSD均小于4.23%。加样回收率在95.35~106.21%范围内，4℃放置24h稳定性良好。芪苈强心复方配伍改变了复方中附子的上述9个生物碱的构成比和总生物碱含量，毒性最大的DDAs（AC、MA、HA）含量明显减少，而MDAs相对含量显著增高。
结论：该方法简便快速，灵敏度高，选择性好，可用于芪苈强心胶囊和附子中9个乌头类生物碱成分的含量测定，并为芪苈强心胶囊的质量控制提供了新的分析方法和手段。
第五部分基于UPLC/Q-TOF-MS/MS的芪苈强心胶囊中9个乌头类生物碱大鼠体内的药代动力学研究
目的：建立一种UPLC/Q-TOF-MS/MS的芪苈强心胶囊中9个乌头类生物碱大鼠体内药代动力学研究分析方法，建立其药-时曲线，获得药动学参数与特征，为临床用药提供参考。
方法：大鼠禁食不禁水12h，灌胃给予芪苈强心胶囊内容物，分别于给药前和给药后0.25、0.5、1、2、4、6、8、10、12、24、36、48h乙醚麻醉腹主动脉取血，至经肝素处理的离心管中，3000r/min离心10min，分离血浆，采用固相萃取法预处理样品，内标为地西泮。PhenomenexKinetex C18100×2.1mm,2.7μm；流动相为0.1%甲酸水溶液(A)-乙腈(B)，梯度洗脱程序为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。柱温：40℃，流速：400μL/min。质谱条件离子化模式：电喷雾离子源（ESI），正离子模式；TOF-MS质谱扫描范围为50-1500Da，MS/MS质谱扫描范围为50-1500Da，喷雾电压（IS）为5500V；雾化温度为550℃，雾化气（Gas1, N2）为50psi，辅助气（Gas2, N2）为50psi，气帘气（Cur, N2）为35psi，裂解电压(DP)60V，碰撞能量（CE）44V，碰撞能量扩展（CES）15V。准确质量数用APCI Positive校正液（利血平）校正。数据获取和处理分析采用Analyst TF1.6软件、 PeakView1.2软件和MultiQuant2.1.1软件。药动学数据采用非室模型处理，用excel软件分析数据。达峰时间(Tmax)和峰浓度(Cmax)由血药浓度曲线直接获得。消除常数（k）由曲线最后4个点的斜率的对数计算得到。消除半衰期T1/2=0.693/k。
结果：血浆中附子灵、尼奥林、塔拉地萨敏、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、乌头碱和次乌头碱在0.25～75ng/mL范围内线性关系良好（r2≥0.9946），最低定量限（LLOQ）≤0.247ng/mL。日内、日间精密度的相对标准偏差（RSD）0.4%~5.0%，相对误差（RE）为-2.4%~6.7%。平均提取回收率为85.70%~97.37%，基质效应分别介于94.63~99.32%。大鼠灌胃芪苈强心胶囊内容物后附子灵、尼奥林、塔拉地萨敏、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、乌头碱和次乌头碱在体内吸收迅速，迅速分散，药时曲线均出现双峰现象，与文献报道相符；消除相比较平缓，我们推测与生物碱类本身代谢特点、复方配伍以及给药剂量高有关。
结论：该法灵敏度高、选择性好、精密度好，可用于大鼠灌胃芪苈强心胶囊后血浆中附子灵、尼奥林、塔拉地萨敏、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、乌头碱和次乌头碱的药动学研究。根据药动学结果由此可推断芪苈强心胶囊复方配伍可能影响活性成分的药动学特征。
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/MS
Objective: 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 qualitycontrol
Conclusion: 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 capsule
Objective：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/MS
Objective: 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 the
Methods: 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 Analysis
Objective: 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 benzoylmesaconine
Methods: 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 method
Objective: 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.
第一部分UPLC/Q-TOF-MS/MS法鉴定芪苈强心胶囊中化学成分
目的：建立一种超高效液相色谱与串联四极杆飞行时间质谱仪联用技术(UPLC/Q-TOF-MS/MS)全面快速地阐明芪苈强心胶囊的化学组成的分析方法。
方法：取芪苈强心胶囊样品研细，取约0.3g，精密称定，置具塞锥形瓶中，加入50%甲醇10mL，超声处理30min，放冷，滤过，用50%甲醇补足至原重量后，摇匀，16000r/min离心10min，取上清夜，过滤（0.22μm），取滤液进样分析。色谱条件：色谱柱：Phenomenex Kinetex C18100×2.1mm,2.6μm。流动相为0.1%甲酸水溶液(A)-乙腈(B)，梯度洗脱程序为：(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。柱温：40℃；流速：400μL/min；进样体积：5μL。质谱色谱条件：离子化模式为电喷雾正、负离子模式，正负离子源电压分别为5500V/-4500V，离子源温度为550℃，裂解电压（DP）分别为60V/-55V，碰撞能量（CE）分别为45eV/-40eV，碰撞能量扩展（CES）分别为15eV/20eV。雾化气体为氮气，辅助气1为55PSI，辅助气2为55PSI，气帘气为35PSI。一级质谱母离子扫描范围为100-1500，IDA设置响应值超过100cps的8个最高峰进行二级质谱扫描，子离子扫描范围为50-1500，开启动态背景扣除（DBS）。数据采集软件：Analyst TF1.6software（AB SCEIX，USA）；数据处理软件系统：Peakview1.2software（AB SCEIX，USA）。获取UPLC/Q-TOF MS/MS全扫描质量色谱图和源内裂解的UPLC-TOF/MS色谱图，参照对照品信息或通过分子离子精确质量和同位素拟合度的分析确定分子式，在数据库中检索匹配的化合物，进而解析源内裂解的质谱图，从而对各色谱峰进行指认。并在相同实验条件下对芪苈强心胶囊及其组方各单味药的色谱图进行比对分析,归属色谱峰来源。
结果：从芪苈强心胶囊色谱图中共鉴定了139个色谱峰，主要成分包括三萜皂苷类、黄酮类、生物碱类、酚酸类、萜类等。本研究比较全面地阐明了芪苈强心胶囊的化学组成，为芪苈强心胶囊的药效物质基础研究和质量控制奠定了基础。
结论：该方法简便、快速，灵敏度，可用于芪苈强心胶囊血中移行成分定性分析。
第二部分芪苈强心胶囊UPLC血清指纹图谱研究
目的：采用超高效液相色谱(UPLC)法建立芪苈强心胶囊血清指纹图谱，为芪苈强心胶囊血清药物化学及药效物质基础研究提供依据。
方法：大鼠灌胃给药制备含药血清，采用Waters ACQUITY UPLCBEH C18色谱柱(2.1mm×100mm，1.7μm)，流动相乙腈-0.1%甲酸水，梯度洗脱，流速0.5mL·min-1，柱温40℃，检测波长280nm，分析芪苈强心胶囊大鼠含药血清，并与相同条件下空白血清、体外全方及其11个单味药图谱比对，对色谱峰归属进行分析。
结果：建立了芪苈强心胶囊的UPLC血清指纹图谱，共标定了13个共有峰，指认了其中4个共有峰，均为药源性成分，来自香加皮。10批芪苈强心胶囊血清指纹图谱的相似度均在0.868～1.000。
结论：本研究建立的方法准确、可靠、重现性好，可用于芪苈强心胶囊血清药物化学及药效物质基础的研究。
第三部分UPLC/Q-TOF-MS/MS法鉴定芪苈强心胶囊血中移行成分
目的：建立一种超高效液相色谱与串联四极杆飞行时间质谱仪联用技术(UPLC/Q-TOF-MS/MS)全面快速地阐明芪苈强心胶囊血中移行成分的分析方法。
方法：采用UPLC/Q-TOF-MS/MS方法，对比芪苈强心胶囊、空白血清、芪苈强心胶囊含药血清总离子流图，质谱图等信息，通过对比各色谱峰的保留时间及质谱图裂解规律，确认大鼠灌胃芪苈强心胶囊内容物后血中移行成分及其归属。
结果：大鼠口服芪苈强心胶囊后血中共发现61个入血成分，其中34个为芪苈强心胶囊中原形成分，27个为原形药物的代谢产物。本研究将为阐明芪苈强心胶囊药效物质基础提供依据。
结论：该方法简便、快速，灵敏度，可以用于芪苈强心胶囊化学成分定性分析。
第四部分UPLC/Q-TOF-MS/MS法快速测定芪苈强心胶囊及其附子中的9个乌头类生物碱成分含量
目的：建立一种简便、准确、可靠的UPLC/Q-TOF-MS/MS分析方法，用于芪苈强心胶囊和附子中9个乌头类生物碱成分的含量测定。
方法：取芪苈强心胶囊样品研细，取约0.3g，精密称定，置具塞锥形瓶中，加入50%甲醇10mL，超声处理30min，放冷，滤过，用50%甲醇补足至原重量后，摇匀，16000r/min离心10min，取上清夜，过滤（0.22μm），取滤液进样分析。色谱条件：色谱柱：Phenomenex Kinetex C18100×2.1mm,2.7μm。流动相由0.1%甲酸水（A）和乙腈（B）组成，梯度洗脱：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).柱温：40℃；流速：400μL/min；进样体积：5μL。每次进样预平衡5min，再进行梯度洗脱。质谱色谱条件：离子喷雾电压，5.5KV；离子源温度,550℃;解簇电压(DP)，60V和碰撞能量(CE)，35eV。氮气为雾化气和辅助气，雾化气50psi，辅助气50psi，气帘气35psi。在m/z100-1500amu质量范围ESI正离子模式下进行全扫描，积累时间250ms。IDA采用标准：每个分析物，超过100cps的八个最强的碎片离子在100~1500amu质量范围内进行子离子扫描，累积时间70ms。碰撞电压差20eV，动态背景扣DBS开启。自动校准系统(CDS)对MS和MS/MS自动进行调谐和校正。数据获取和处理分析采用Analyst TF1.6软件、PeakView1.2软件和MultiQuant2.1.1软件。
结果：在一定浓度区间内9种分析物的线性关系良好，r2>0.9940；所有分析物定量限LOQ范围0.03~0.50ng/mL，日内精密度、日间精密度RSD均小于3.38%，重复性试验RSD均小于4.23%。加样回收率在95.35~106.21%范围内，4℃放置24h稳定性良好。芪苈强心复方配伍改变了复方中附子的上述9个生物碱的构成比和总生物碱含量，毒性最大的DDAs（AC、MA、HA）含量明显减少，而MDAs相对含量显著增高。
结论：该方法简便快速，灵敏度高，选择性好，可用于芪苈强心胶囊和附子中9个乌头类生物碱成分的含量测定，并为芪苈强心胶囊的质量控制提供了新的分析方法和手段。
第五部分基于UPLC/Q-TOF-MS/MS的芪苈强心胶囊中9个乌头类生物碱大鼠体内的药代动力学研究
目的：建立一种UPLC/Q-TOF-MS/MS的芪苈强心胶囊中9个乌头类生物碱大鼠体内药代动力学研究分析方法，建立其药-时曲线，获得药动学参数与特征，为临床用药提供参考。
方法：大鼠禁食不禁水12h，灌胃给予芪苈强心胶囊内容物，分别于给药前和给药后0.25、0.5、1、2、4、6、8、10、12、24、36、48h乙醚麻醉腹主动脉取血，至经肝素处理的离心管中，3000r/min离心10min，分离血浆，采用固相萃取法预处理样品，内标为地西泮。PhenomenexKinetex C18100×2.1mm,2.7μm；流动相为0.1%甲酸水溶液(A)-乙腈(B)，梯度洗脱程序为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。柱温：40℃，流速：400μL/min。质谱条件离子化模式：电喷雾离子源（ESI），正离子模式；TOF-MS质谱扫描范围为50-1500Da，MS/MS质谱扫描范围为50-1500Da，喷雾电压（IS）为5500V；雾化温度为550℃，雾化气（Gas1, N2）为50psi，辅助气（Gas2, N2）为50psi，气帘气（Cur, N2）为35psi，裂解电压(DP)60V，碰撞能量（CE）44V，碰撞能量扩展（CES）15V。准确质量数用APCI Positive校正液（利血平）校正。数据获取和处理分析采用Analyst TF1.6软件、 PeakView1.2软件和MultiQuant2.1.1软件。药动学数据采用非室模型处理，用excel软件分析数据。达峰时间(Tmax)和峰浓度(Cmax)由血药浓度曲线直接获得。消除常数（k）由曲线最后4个点的斜率的对数计算得到。消除半衰期T1/2=0.693/k。
结果：血浆中附子灵、尼奥林、塔拉地萨敏、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、乌头碱和次乌头碱在0.25～75ng/mL范围内线性关系良好（r2≥0.9946），最低定量限（LLOQ）≤0.247ng/mL。日内、日间精密度的相对标准偏差（RSD）0.4%~5.0%，相对误差（RE）为-2.4%~6.7%。平均提取回收率为85.70%~97.37%，基质效应分别介于94.63~99.32%。大鼠灌胃芪苈强心胶囊内容物后附子灵、尼奥林、塔拉地萨敏、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、乌头碱和次乌头碱在体内吸收迅速，迅速分散，药时曲线均出现双峰现象，与文献报道相符；消除相比较平缓，我们推测与生物碱类本身代谢特点、复方配伍以及给药剂量高有关。
结论：该法灵敏度高、选择性好、精密度好，可用于大鼠灌胃芪苈强心胶囊后血浆中附子灵、尼奥林、塔拉地萨敏、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、乌头碱和次乌头碱的药动学研究。根据药动学结果由此可推断芪苈强心胶囊复方配伍可能影响活性成分的药动学特征。
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/MS
Objective: 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 qualitycontrol
Conclusion: 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 capsule
Objective：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/MS
Objective: 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 the
Methods: 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 Analysis
Objective: 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 benzoylmesaconine
Methods: 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 method
Objective: 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.
引文
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