基于液质联用技术的知母及其复方制剂中多组分同时分析与药物代谢动力学研究
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摘要
知母是百合科植物知母（Anemarrhena asphodeloides Bge.）的干燥根茎，为中国药典收载的常用中药之一，味甘、苦，性寒，入肺、胃、肾经，具有清热泻火、生津润燥之功效。其化学成分主要有甾体皂苷、双苯吡酮类、木脂素类、有机酸类、生物碱、黄酮类、多糖类、微量元素及大量的黏液质等。具有抗炎、抗菌及镇痛解热作用以及抗衰老、抗血小板聚集、降血糖作用。目前知母及其复方制剂已广泛的用于临床。本文采用LC-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，对知母中的化合物进行定量分析，并利用化学计量学的方法对其质量进行有效合理的控制。同时研究了知母贝母复方制剂（二母制剂）的6种成分在动物体内的药代动力学过程和排泄规律，为进一步合理使用中药复方提供理论基础。
第一部分HPLC-MS法结合化学计量学分析测定知母中的9种成分
目的：建立一种快速、准确、可同时测定知母中9种化学成分（4种甾体皂苷成分、2种双苯吡酮、2种异黄酮及1个蒽醌类化合物）的HPLC-ESI-MS分析方法，并采用化学计量学分析对不同来源知母与知母的不同部位的质量进行有效合理的分析。
方法：取知母药材粉末约1.0g，精密称定，加入30mL乙醇，密塞，称定重量，超声处理60min，放冷，称重，用乙醇补足至原重量后，摇匀，过滤（0.45m微孔滤膜），取续滤液，10μL进样后测定。含量测定方法的建立在MRM正负离子扫描模式下进行，并对30批不同来源的知母和知母不同部位进行测定分析，聚类分析和主成分分析对30批样品的含量测定结果进行区分和分类。质谱色谱条件：电喷雾离子源（ESI）；正负离子在同一周期内进行转换同时监测；源喷射电压（正离子）5500V；（负离子）-4500V。离子源温度：600℃。雾化气（Gas1）：40psi，加热气（Gas2）：50psi，帘气：25psi。色谱条件：色谱柱： Diamonsil C18column（250mm×4.6mm，5μm）；柱温：30℃；洗脱程序：0-2.5min，流动相乙腈-0.1%甲酸水(5:95, v/v)线性变至乙腈-0.1%甲酸水(55:45, v/v)；2.5-5min，流动相乙腈-0.1%甲酸水(55:45, v/v)线性变至为乙腈-0.1%甲酸水(95:5, v/v)；5-20min，乙腈-0.1%甲酸水(95:5,v/v)下等度洗脱；最后流动相的比例迅速转变为最初的乙腈-0.1%甲酸水(5:95, v/v)。每次进样前要预平衡6min，再进行梯度洗脱。运行时间：20min；流速：800μL/min，进样量10μL。
结果：在一定浓度区间内9种活性成分的线性关系良好，r~2>0.9912；日内和日间精密度分别小于2.21%和2.34%；平均回收率96.8%~103.9%，RSD0.81to2.32%；样品储存于4℃，48h内稳定性良好(RSD <2.28%)。30批样品含量采用上述HPLC-ESI-MS方法测得。同一周期内通过转换正负离子源20min内可完成一次分析。MRM正负离子扫描模式降低了噪音水平，提高了分析的灵敏度。30批样品总含量范围为23.01到132.65mg/g，含量差异较大。HB-4，HB-5和HB-6含量最高(132.65,120.59, and116.92mg/g)；而HBL-1, HBL-2和HBL-3含量最低(23.01,34.09, and29.68mg/g)。30批样品总含量平均值90.46mg/g。6批样品（HBP-1,-2,-3andHBS-1,-2,-3）略低于平均总含量，HBL-1,-2,-3总含量最低。知母皂苷BII是样品中含量最高的活性成分43.79mg/g，其次是芒果苷36.13mg/g。主成分分析及聚类分析对30批知母药材成功分类区分，证明了道地药材质量最佳。知母地上地下部分均含有活性成分，并且含量较高，也应得到充分的开发利用。
结论：该方法简便快速，灵敏度高，选择性好，为知母药材的质量控制提供了新的方法和手段。
第二部分HPLC-MS法同时测定大鼠口服知母、贝母与复方提取物后血浆中6种成分及其药动学比较
目的：二母制剂，用于治疗哮喘和支气管炎症的传统中药复方制剂，最早记载于我国古代的《景岳全书》和《急救仙方》等著名方剂学中医古籍中。该制剂不仅在中国有悠久的历史，在东方国家如韩国也被广泛使用治疗咳嗽和哮喘。二母制剂由知母和贝母两种药材等比例组合后提取制备。新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ是二母制剂中的主要活性成分。该研究建立一种基于多组分同时测定大鼠血浆中新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ含量的HPLC-MS方法，并用于大鼠灌胃给予知母、贝母单味药材提取物和知母、贝母混合提取物后该6种成分的药代动力学研究，建立其药-时曲线，阐明各自药动学参数与特征，并比较了单方与复方制剂的药动学差异，考察了配伍对活性成分药动学的影响。
方法：知母（100g）、贝母（100g）粉碎后分别用70%乙醇按1:10,1:10,1:5的比例回流提取3次后浓缩至2g/mL的提取物。知母和贝母提取物按1：1比例混合后制成二母制剂。知母和贝母提取物中新芒果苷、芒果苷、知母皂苷BII、知母皂苷AⅢ、含量分别为8.76，21.16，74.24，4.16，0.32和0.57mg/mL。
大鼠随机分为3组，分别灌胃给予单味知母或贝母提取物，知母、贝母混合提取物后，由眼眦静脉丛取血，取血时间为5,10,15,30,60,120,180,240,360,480,600,1440,2160和2880min。样品置于肝素化的试管内，离心得到上清液为血浆样品，采用沉淀蛋白法预处理样品，内标为磺胺甲噁唑。C18色谱柱，洗脱程序0-7.0min，流动相乙腈-0.1%甲酸水(5:95,v/v)线性变至乙腈-0.1%甲酸水(55:45, v/v)；7.0-10.0min，流动相乙腈-0.1%甲酸水(55:45, v/v)线性变至乙腈-0.1%甲酸水(95:5, v/v)；10.0-15.0min，乙腈-0.1%甲酸水(95:5,v/v)下等度洗脱；最后10.0-15.2min流动相的比例迅速转变为最初的乙腈-0.1%甲酸水(5:95, v/v)。每次进样前要预平衡6min，再进行梯度洗脱。流速：800μL/min，进样量10μL。离子源为电喷雾（ESI），正负离子多周期同时扫描，多反应模式监测（MRM）进行定量。监测分为3个阶段，0-6.19min在负离子模式监测新芒果苷和芒果苷；6.19-6.22min由负离子模式转换成正离子模式；之后在正离子模式下监测知母皂苷BII、知母皂苷AⅢ、贝母素甲、贝母素乙和内标。6种被测成分的监测离子对分别为新芒果苷m/z583.0/331.0，芒果苷m/z421.1/301.0，贝母素甲m/z432.5/414.5，贝母素乙m/z430.5/412.5，知母皂苷BII m/z903.1/417.2，知母皂苷AⅢ m/z741.4/417.3和磺胺甲噁唑m/z254.0/156.0。
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：血浆中新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ分别在4.63～1852.8、2.92～1168、3.56～1424、4.44～1776、4.40～1760和4.00～1600ng/mL范围内线性关系良好（r~2≥0.9946），最低定量限（LLOQ）≤2.27ng/mL。日内、日间精密度的相对标准偏差（RSD）均小于7.8%，相对误差（RE）为-7.4%～7.4%。平均提取回收率为80.6%～102.3%。大鼠灌胃知母或贝母单味提取物和混合提取物后新芒果苷、芒果苷、贝母素甲和贝母素乙的药动学参数有显著性差异(P<0.05)。与灌胃知母提取物相比，复方提取物大鼠的芒果苷和新芒果苷的Cmax和AUC均显著增加，T1/2有所下降，Tmax无显著性差异。灌胃知母和灌胃复方制剂后，知母皂苷BII和知母皂苷AⅢ的药动学参数没有显著性差异。与灌胃贝母提取物相比，灌胃复方提取物大鼠的贝母素甲和贝母素乙的Cmax和AUC都显著增加，T1/2明显延长，Tmax无显著性差异。
结论：该法灵敏度高、选择性好、精密度好，节约时间和试剂，可用于大鼠灌胃提取物后血浆中新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ的药动学研究。根据药动学结果由此可推断二者配伍可影响活性成分的药动学特征，并可以初步推断知母和贝母配伍的合理性。
第三部分HPLC-MS法同时测定大鼠口服知母、贝母与复方提取物后胆汁和尿液中6种成分及其在胆汁和尿液中的排泄动力学
目的：二母制剂，用于治疗哮喘和支气管炎症的传统中药复方制剂，最早记载于我国古代的《景岳全书》和《急救仙方》等著名方剂学中医古籍中。该制剂不仅在中国有悠久的历史，在东方国家如韩国也被广泛使用治疗咳嗽和哮喘。二母制剂由知母和贝母两种药材等比例组合后提取制备。新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ是二母制剂中的主要活性成分。本研究建立一种可同时测定大鼠胆汁和尿液中6种成分——新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ含量的HPLC-MS方法，并用于大鼠灌胃给予单味知母或贝母药材提取物和二母混合药材提取物后，该6种成分的胆汁和尿液排泄动力学研究。通过比较口服不同提取物后的排泄动力学参数，考察其配伍对活性成分的排泄动力学参数的影响。
方法：健康SD雄性大鼠18只，随机分为3组，每组6只，大鼠分别灌胃给予单味知母或贝母药材提取物和知母贝母混合药材提取物，麻醉后行胆管切开术，分别在2,4,6,8,10,24,36和48h收集胆汁样品。另取
SD大鼠18只，随机分为3组，每组6只，大鼠分别灌胃给予单味知母或贝母药材提取物和二母混合药材提取物，收集0–4,4–8,8–12,12–24,24–36,36–48,48–60和60–72h尿液。采用直接进样的方法进行样品前处理，内标为SMZ。C18柱，洗脱程序0-7.0min，流动相乙腈-0.1%甲酸水(5:95, v/v)线性变至乙腈-0.1%甲酸水(55:45, v/v)；7.0-10.0min，流动相乙腈-0.1%甲酸水(55:45, v/v)线性变至为乙腈-0.1%甲酸水(95:5, v/v)；10.0-15.0min，乙腈-0.1%甲酸水(95:5,v/v)下等度洗脱；最后10.0-15.2min流动相的比例迅速转变为最初的乙腈-0.1%甲酸水(5:95, v/v)。每次进样前要预平衡6min，再进行梯度洗脱。流速：800μL/min，进样量10μL。离子源为电喷雾（ESI），正负离子多周期同时扫描，多反应模式监测（MRM）。监测分为3个阶段，0-6.19min在负离子模式监测新芒果苷和芒果苷；6.19-6.22min由负离子模式转换成正离子模式；之后在正离子模式下监测知母皂苷BII、知母皂苷AⅢ、贝母素甲、贝母素乙和内标。6种被测成分的监测离子对分别为新芒果苷m/z583.0/331.0，芒果苷m/z421.1/301.0，贝母素甲m/z432.5/414.5，贝母素乙m/z430.5/412.5，知母皂苷BII m/z903.1/417.2，知母皂苷A Ⅲ m/z741.4/417.3和磺胺甲噁唑m/z254.0/156.0。测定大鼠灌胃给予不同提取物后胆汁和尿液中6种化学成分累积排泄率。
结果：结果表明，所测的6种活性成分仅有不足7%的原型从尿液中排出；不足5%的原型从胆汁排泄。灌胃知母贝母复方提取物和单方提取物后该6种成分表现出显著不同的排泄行为。灌胃复方提取物后6种化合物在尿液中的排泄水平均高于单方提取物的排泄水平（约4倍到14倍）。而在胆汁排泄中，灌胃复方提取物后只有芒果苷、贝母素甲和贝母素乙的排泄水平高于单方提取物，其余3种成分新芒果苷、知母皂苷BII和知母皂苷AⅢ的排泄水平均低于单方提取物。
结论：经方法学考察符合生物样品的测定要求，可用于大鼠胆汁和尿液中6种成分浓度的测定及排泄研究。无论口服知母贝母复方提取物还是单方提取物待测的6种成分在体内都经过了广泛的代谢或生物转化。不同的代谢机制导致了不同的排泄行为。
第四部分HPLC-MS法同时测定金莲清热颗粒中7种成分的含量
目的：建立HPLC-MS法测定金莲清热颗粒中荭草苷，牡荆苷，金丝桃苷，新芒果苷，芒果苷，知母皂苷BⅡ和知母皂苷AⅢ含量的方法。
方法：取金莲清热颗粒，研细，精密称定粉末0.500g，置锥形瓶中，精密加入乙腈50mL，称定质量，超声(功率:100W；频率:25kHz)处理1h，放冷，再称定质量，用乙腈补充所失的重量，摇匀，离心，0.45μm滤膜滤过，续滤液作为供试品溶液，稀释20倍后进样10μL。
质谱色谱条件：MRM正负离子扫描模式、电喷雾离子源（ESI）；正负离子在同一周期内进行转换同时监测；源喷射电压（正离子）5500V；（负离子）-4500V。离子源温度：600℃。雾化气（Gas1）：40psi，加热气（Gas2）：50psi，帘气：25psi。色谱柱：C18Diamonsil column（250mm×4.6mm，5μm）；柱温：30℃；洗脱程序：0-2.5min，流动相乙腈-0.1%甲酸水(5:95, v/v)线性变至乙腈-0.1%甲酸水(55:45, v/v)；2.5-5min，流动相乙腈-0.1%甲酸水(55:45, v/v)线性变至为乙腈-0.1%甲酸水(95:5, v/v)；5-20min，乙腈-0.1%甲酸水(95:5,v/v)下等度洗脱；最后流动相的比例迅速转变为最初的乙腈-0.1%甲酸水(5:95, v/v)。每次进样前要预平衡6min，再进行梯度洗脱。运行时间：20min；流速：800μL/min，进样量10μL。
结果：在15min内金莲清热颗粒中7种主要成分峰面积与浓度呈良好的线性关系；加样回收率分别为99.34%,99.28%,101.6%,101.3%,100.5%,101.9%和98.86%(n=3)。RSD值分别为1.12%,1.36%,1.57%,1.74%,1.52%,1.43%和0.92%。
结论：该方法简便，准确，重现性好，专属性高，可用于金莲清热颗粒的质量控制。
第五部分HPLC-MS法同时测定知柏地黄丸中9种成分的含量
目的：建立HPLC-MS法测定知柏地黄浓缩丸中新芒果苷，芒果苷，知母皂苷BⅡ，知母皂苷AⅢ，小檗碱，莫诺苷，马钱苷，芍药苷和丹皮酚含量的方法。
方法：取知柏地黄丸适量，研细，精密称定粉末0.4133g，置锥形瓶中，精密加入50%乙腈50mL，称定质量，超声处理(功率:100W；频率:25kHz)0.5h，放冷，再称定质量，用50%乙腈补足减失的重量，摇匀，离心，0.45μm滤膜滤过，溶液稀释20倍后取10μL进样分析。
采用Diamonsil C18柱(150mm×4.6mm,5μm)；柱温30℃；乙腈-0.1%甲酸水溶液为流动相以800μL/min梯度洗脱；质谱条件：采用电喷雾离子源正负离子模式同时进行检测，多反应监测模式(MRM)用于定量测定。
结果：在15min内知柏地黄丸中9个有效成分峰面积与浓度线性关系良好；加样回收率(n=3)分别为97.4%，99.2%，100.3%，101.8%，99.5%，101.6%，99.6%，101.7%和98.86%。RSD值分别为1.2%，1.6%，1.0%，0.7%，1.5%，0.4%，1.0%，1.1%和0.9%。
结论：该方法简便，准确，重现性好，专属性高，可用于知柏地黄丸的质量控制。
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 techniques
Objective: 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 method
Objective: 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 study
Objective: 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-MS
Objective: 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-MS
Objective: 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.
第一部分HPLC-MS法结合化学计量学分析测定知母中的9种成分
目的：建立一种快速、准确、可同时测定知母中9种化学成分（4种甾体皂苷成分、2种双苯吡酮、2种异黄酮及1个蒽醌类化合物）的HPLC-ESI-MS分析方法，并采用化学计量学分析对不同来源知母与知母的不同部位的质量进行有效合理的分析。
方法：取知母药材粉末约1.0g，精密称定，加入30mL乙醇，密塞，称定重量，超声处理60min，放冷，称重，用乙醇补足至原重量后，摇匀，过滤（0.45m微孔滤膜），取续滤液，10μL进样后测定。含量测定方法的建立在MRM正负离子扫描模式下进行，并对30批不同来源的知母和知母不同部位进行测定分析，聚类分析和主成分分析对30批样品的含量测定结果进行区分和分类。质谱色谱条件：电喷雾离子源（ESI）；正负离子在同一周期内进行转换同时监测；源喷射电压（正离子）5500V；（负离子）-4500V。离子源温度：600℃。雾化气（Gas1）：40psi，加热气（Gas2）：50psi，帘气：25psi。色谱条件：色谱柱： Diamonsil C18column（250mm×4.6mm，5μm）；柱温：30℃；洗脱程序：0-2.5min，流动相乙腈-0.1%甲酸水(5:95, v/v)线性变至乙腈-0.1%甲酸水(55:45, v/v)；2.5-5min，流动相乙腈-0.1%甲酸水(55:45, v/v)线性变至为乙腈-0.1%甲酸水(95:5, v/v)；5-20min，乙腈-0.1%甲酸水(95:5,v/v)下等度洗脱；最后流动相的比例迅速转变为最初的乙腈-0.1%甲酸水(5:95, v/v)。每次进样前要预平衡6min，再进行梯度洗脱。运行时间：20min；流速：800μL/min，进样量10μL。
结果：在一定浓度区间内9种活性成分的线性关系良好，r~2>0.9912；日内和日间精密度分别小于2.21%和2.34%；平均回收率96.8%~103.9%，RSD0.81to2.32%；样品储存于4℃，48h内稳定性良好(RSD <2.28%)。30批样品含量采用上述HPLC-ESI-MS方法测得。同一周期内通过转换正负离子源20min内可完成一次分析。MRM正负离子扫描模式降低了噪音水平，提高了分析的灵敏度。30批样品总含量范围为23.01到132.65mg/g，含量差异较大。HB-4，HB-5和HB-6含量最高(132.65,120.59, and116.92mg/g)；而HBL-1, HBL-2和HBL-3含量最低(23.01,34.09, and29.68mg/g)。30批样品总含量平均值90.46mg/g。6批样品（HBP-1,-2,-3andHBS-1,-2,-3）略低于平均总含量，HBL-1,-2,-3总含量最低。知母皂苷BII是样品中含量最高的活性成分43.79mg/g，其次是芒果苷36.13mg/g。主成分分析及聚类分析对30批知母药材成功分类区分，证明了道地药材质量最佳。知母地上地下部分均含有活性成分，并且含量较高，也应得到充分的开发利用。
结论：该方法简便快速，灵敏度高，选择性好，为知母药材的质量控制提供了新的方法和手段。
第二部分HPLC-MS法同时测定大鼠口服知母、贝母与复方提取物后血浆中6种成分及其药动学比较
目的：二母制剂，用于治疗哮喘和支气管炎症的传统中药复方制剂，最早记载于我国古代的《景岳全书》和《急救仙方》等著名方剂学中医古籍中。该制剂不仅在中国有悠久的历史，在东方国家如韩国也被广泛使用治疗咳嗽和哮喘。二母制剂由知母和贝母两种药材等比例组合后提取制备。新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ是二母制剂中的主要活性成分。该研究建立一种基于多组分同时测定大鼠血浆中新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ含量的HPLC-MS方法，并用于大鼠灌胃给予知母、贝母单味药材提取物和知母、贝母混合提取物后该6种成分的药代动力学研究，建立其药-时曲线，阐明各自药动学参数与特征，并比较了单方与复方制剂的药动学差异，考察了配伍对活性成分药动学的影响。
方法：知母（100g）、贝母（100g）粉碎后分别用70%乙醇按1:10,1:10,1:5的比例回流提取3次后浓缩至2g/mL的提取物。知母和贝母提取物按1：1比例混合后制成二母制剂。知母和贝母提取物中新芒果苷、芒果苷、知母皂苷BII、知母皂苷AⅢ、含量分别为8.76，21.16，74.24，4.16，0.32和0.57mg/mL。
大鼠随机分为3组，分别灌胃给予单味知母或贝母提取物，知母、贝母混合提取物后，由眼眦静脉丛取血，取血时间为5,10,15,30,60,120,180,240,360,480,600,1440,2160和2880min。样品置于肝素化的试管内，离心得到上清液为血浆样品，采用沉淀蛋白法预处理样品，内标为磺胺甲噁唑。C18色谱柱，洗脱程序0-7.0min，流动相乙腈-0.1%甲酸水(5:95,v/v)线性变至乙腈-0.1%甲酸水(55:45, v/v)；7.0-10.0min，流动相乙腈-0.1%甲酸水(55:45, v/v)线性变至乙腈-0.1%甲酸水(95:5, v/v)；10.0-15.0min，乙腈-0.1%甲酸水(95:5,v/v)下等度洗脱；最后10.0-15.2min流动相的比例迅速转变为最初的乙腈-0.1%甲酸水(5:95, v/v)。每次进样前要预平衡6min，再进行梯度洗脱。流速：800μL/min，进样量10μL。离子源为电喷雾（ESI），正负离子多周期同时扫描，多反应模式监测（MRM）进行定量。监测分为3个阶段，0-6.19min在负离子模式监测新芒果苷和芒果苷；6.19-6.22min由负离子模式转换成正离子模式；之后在正离子模式下监测知母皂苷BII、知母皂苷AⅢ、贝母素甲、贝母素乙和内标。6种被测成分的监测离子对分别为新芒果苷m/z583.0/331.0，芒果苷m/z421.1/301.0，贝母素甲m/z432.5/414.5，贝母素乙m/z430.5/412.5，知母皂苷BII m/z903.1/417.2，知母皂苷AⅢ m/z741.4/417.3和磺胺甲噁唑m/z254.0/156.0。
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：血浆中新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ分别在4.63～1852.8、2.92～1168、3.56～1424、4.44～1776、4.40～1760和4.00～1600ng/mL范围内线性关系良好（r~2≥0.9946），最低定量限（LLOQ）≤2.27ng/mL。日内、日间精密度的相对标准偏差（RSD）均小于7.8%，相对误差（RE）为-7.4%～7.4%。平均提取回收率为80.6%～102.3%。大鼠灌胃知母或贝母单味提取物和混合提取物后新芒果苷、芒果苷、贝母素甲和贝母素乙的药动学参数有显著性差异(P<0.05)。与灌胃知母提取物相比，复方提取物大鼠的芒果苷和新芒果苷的Cmax和AUC均显著增加，T1/2有所下降，Tmax无显著性差异。灌胃知母和灌胃复方制剂后，知母皂苷BII和知母皂苷AⅢ的药动学参数没有显著性差异。与灌胃贝母提取物相比，灌胃复方提取物大鼠的贝母素甲和贝母素乙的Cmax和AUC都显著增加，T1/2明显延长，Tmax无显著性差异。
结论：该法灵敏度高、选择性好、精密度好，节约时间和试剂，可用于大鼠灌胃提取物后血浆中新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ的药动学研究。根据药动学结果由此可推断二者配伍可影响活性成分的药动学特征，并可以初步推断知母和贝母配伍的合理性。
第三部分HPLC-MS法同时测定大鼠口服知母、贝母与复方提取物后胆汁和尿液中6种成分及其在胆汁和尿液中的排泄动力学
目的：二母制剂，用于治疗哮喘和支气管炎症的传统中药复方制剂，最早记载于我国古代的《景岳全书》和《急救仙方》等著名方剂学中医古籍中。该制剂不仅在中国有悠久的历史，在东方国家如韩国也被广泛使用治疗咳嗽和哮喘。二母制剂由知母和贝母两种药材等比例组合后提取制备。新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ是二母制剂中的主要活性成分。本研究建立一种可同时测定大鼠胆汁和尿液中6种成分——新芒果苷、芒果苷、贝母素甲、贝母素乙、知母皂苷BII和知母皂苷AⅢ含量的HPLC-MS方法，并用于大鼠灌胃给予单味知母或贝母药材提取物和二母混合药材提取物后，该6种成分的胆汁和尿液排泄动力学研究。通过比较口服不同提取物后的排泄动力学参数，考察其配伍对活性成分的排泄动力学参数的影响。
方法：健康SD雄性大鼠18只，随机分为3组，每组6只，大鼠分别灌胃给予单味知母或贝母药材提取物和知母贝母混合药材提取物，麻醉后行胆管切开术，分别在2,4,6,8,10,24,36和48h收集胆汁样品。另取
SD大鼠18只，随机分为3组，每组6只，大鼠分别灌胃给予单味知母或贝母药材提取物和二母混合药材提取物，收集0–4,4–8,8–12,12–24,24–36,36–48,48–60和60–72h尿液。采用直接进样的方法进行样品前处理，内标为SMZ。C18柱，洗脱程序0-7.0min，流动相乙腈-0.1%甲酸水(5:95, v/v)线性变至乙腈-0.1%甲酸水(55:45, v/v)；7.0-10.0min，流动相乙腈-0.1%甲酸水(55:45, v/v)线性变至为乙腈-0.1%甲酸水(95:5, v/v)；10.0-15.0min，乙腈-0.1%甲酸水(95:5,v/v)下等度洗脱；最后10.0-15.2min流动相的比例迅速转变为最初的乙腈-0.1%甲酸水(5:95, v/v)。每次进样前要预平衡6min，再进行梯度洗脱。流速：800μL/min，进样量10μL。离子源为电喷雾（ESI），正负离子多周期同时扫描，多反应模式监测（MRM）。监测分为3个阶段，0-6.19min在负离子模式监测新芒果苷和芒果苷；6.19-6.22min由负离子模式转换成正离子模式；之后在正离子模式下监测知母皂苷BII、知母皂苷AⅢ、贝母素甲、贝母素乙和内标。6种被测成分的监测离子对分别为新芒果苷m/z583.0/331.0，芒果苷m/z421.1/301.0，贝母素甲m/z432.5/414.5，贝母素乙m/z430.5/412.5，知母皂苷BII m/z903.1/417.2，知母皂苷A Ⅲ m/z741.4/417.3和磺胺甲噁唑m/z254.0/156.0。测定大鼠灌胃给予不同提取物后胆汁和尿液中6种化学成分累积排泄率。
结果：结果表明，所测的6种活性成分仅有不足7%的原型从尿液中排出；不足5%的原型从胆汁排泄。灌胃知母贝母复方提取物和单方提取物后该6种成分表现出显著不同的排泄行为。灌胃复方提取物后6种化合物在尿液中的排泄水平均高于单方提取物的排泄水平（约4倍到14倍）。而在胆汁排泄中，灌胃复方提取物后只有芒果苷、贝母素甲和贝母素乙的排泄水平高于单方提取物，其余3种成分新芒果苷、知母皂苷BII和知母皂苷AⅢ的排泄水平均低于单方提取物。
结论：经方法学考察符合生物样品的测定要求，可用于大鼠胆汁和尿液中6种成分浓度的测定及排泄研究。无论口服知母贝母复方提取物还是单方提取物待测的6种成分在体内都经过了广泛的代谢或生物转化。不同的代谢机制导致了不同的排泄行为。
第四部分HPLC-MS法同时测定金莲清热颗粒中7种成分的含量
目的：建立HPLC-MS法测定金莲清热颗粒中荭草苷，牡荆苷，金丝桃苷，新芒果苷，芒果苷，知母皂苷BⅡ和知母皂苷AⅢ含量的方法。
方法：取金莲清热颗粒，研细，精密称定粉末0.500g，置锥形瓶中，精密加入乙腈50mL，称定质量，超声(功率:100W；频率:25kHz)处理1h，放冷，再称定质量，用乙腈补充所失的重量，摇匀，离心，0.45μm滤膜滤过，续滤液作为供试品溶液，稀释20倍后进样10μL。
质谱色谱条件：MRM正负离子扫描模式、电喷雾离子源（ESI）；正负离子在同一周期内进行转换同时监测；源喷射电压（正离子）5500V；（负离子）-4500V。离子源温度：600℃。雾化气（Gas1）：40psi，加热气（Gas2）：50psi，帘气：25psi。色谱柱：C18Diamonsil column（250mm×4.6mm，5μm）；柱温：30℃；洗脱程序：0-2.5min，流动相乙腈-0.1%甲酸水(5:95, v/v)线性变至乙腈-0.1%甲酸水(55:45, v/v)；2.5-5min，流动相乙腈-0.1%甲酸水(55:45, v/v)线性变至为乙腈-0.1%甲酸水(95:5, v/v)；5-20min，乙腈-0.1%甲酸水(95:5,v/v)下等度洗脱；最后流动相的比例迅速转变为最初的乙腈-0.1%甲酸水(5:95, v/v)。每次进样前要预平衡6min，再进行梯度洗脱。运行时间：20min；流速：800μL/min，进样量10μL。
结果：在15min内金莲清热颗粒中7种主要成分峰面积与浓度呈良好的线性关系；加样回收率分别为99.34%,99.28%,101.6%,101.3%,100.5%,101.9%和98.86%(n=3)。RSD值分别为1.12%,1.36%,1.57%,1.74%,1.52%,1.43%和0.92%。
结论：该方法简便，准确，重现性好，专属性高，可用于金莲清热颗粒的质量控制。
第五部分HPLC-MS法同时测定知柏地黄丸中9种成分的含量
目的：建立HPLC-MS法测定知柏地黄浓缩丸中新芒果苷，芒果苷，知母皂苷BⅡ，知母皂苷AⅢ，小檗碱，莫诺苷，马钱苷，芍药苷和丹皮酚含量的方法。
方法：取知柏地黄丸适量，研细，精密称定粉末0.4133g，置锥形瓶中，精密加入50%乙腈50mL，称定质量，超声处理(功率:100W；频率:25kHz)0.5h，放冷，再称定质量，用50%乙腈补足减失的重量，摇匀，离心，0.45μm滤膜滤过，溶液稀释20倍后取10μL进样分析。
采用Diamonsil C18柱(150mm×4.6mm,5μm)；柱温30℃；乙腈-0.1%甲酸水溶液为流动相以800μL/min梯度洗脱；质谱条件：采用电喷雾离子源正负离子模式同时进行检测，多反应监测模式(MRM)用于定量测定。
结果：在15min内知柏地黄丸中9个有效成分峰面积与浓度线性关系良好；加样回收率(n=3)分别为97.4%，99.2%，100.3%，101.8%，99.5%，101.6%，99.6%，101.7%和98.86%。RSD值分别为1.2%，1.6%，1.0%，0.7%，1.5%，0.4%，1.0%，1.1%和0.9%。
结论：该方法简便，准确，重现性好，专属性高，可用于知柏地黄丸的质量控制。
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 techniques
Objective: 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 method
Objective: 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 study
Objective: 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-MS
Objective: 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-MS
Objective: 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.
引文
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5LIN Q F, FENG S Q, LI Y L, et al. Study on the antibacterial and antiviralactivity compositions of Trollius chinensis Bunge. Journal of ZhejiangUniversity(Science Edition),2004，31（4）：412-415
6Y.G. Sun, Y.F. Du, Y. Liu, L. Chang, Y.P. Ren, L. Cao, Q. Sun, X.W. Shi, Q.Wang, L.T. Zhang, Simultaneous determination of9components inAnemarrhena asphodeloides by liquid chromatography-tandem massspectrometry combined with chemometric techniques, J Sep Sci.35(2012)1796-1807
1国家药典委员会．中华人民共和国药典:2010年版一部［S］．北京:中国医药科技出版社，2010:820-821
2李明权.知柏地黄丸源流考.中医研究，1997，10(5)：56-57
3陈岳蓉，刘海青.高效液相色谱法测定知柏地黄丸中芒果苷的含量.中国医院药学杂志，2008，28（8）：677-679
4陈骁勇.HPLC法测定知柏地黄丸中菝葜皂苷元的含量.中国药房，2011，22（40）：3826-3827
5刘义梅.薄层色谱法测定知柏地黄丸中丹皮酚的含量.湖北中医杂志，2005，27（7）：52-54
6徐桂香，杨婕，陈钟文.HPLC法测定知柏地黄丸浓缩丸中马钱苷的含量.河南中医学院学报，2007，22（4）：30
7洪菁.HPLC法测定知柏地黄丸浓缩丸中丹皮酚的含量.海峡药学，2008，20（8）：31-33
8陈庆辉，唐元军.知柏地黄丸中盐酸小檗碱和盐酸巴马汀的含量测定.药物分析杂志，2012，32(12)：2198-2202
9翁晓明，李智林.高效液相色谱法测定知柏地黄丸中盐酸小檗碱和丹皮酚含量.中国医院药学杂志2001,21(1)：21-22
10Y.G. Sun, Y.F. Du, Y. Liu, L. Chang, Y.P. Ren, L. Cao, Q. Sun, X.W. Shi, Q.Wang, L.T. Zhang, Simultaneous determination of9components inAnemarrhena asphodeloides by liquid chromatography-tandem massspectrometry combined with chemometric techniques, J Sep Sci.2012，35：1796-1807
11易博，孙赫，原源，等．RP-HPLC法测定知母黄柏药对中新芒果苷、芒果苷和盐酸小檗碱．中草药，2007，38(6):856-858
12劳凤云,李小娜,张卫国,等．山茱萸总苷中莫诺苷及马钱苷HPLC法同时测定．中国公共卫生，2009，25（7）：889-891
13杨正生，彭振辉，姚青海，等．丹皮酚的药理作用研究进展．中国药物与临床，2011，11(5):545-547
1国家药典委员会．中华人民共和国药典（一部）[S].2010年版.北京：中国医药科技出版社，2010：194
2倪梁红，秦民坚．知母资源化学及药理研究进展．中国野生植物资源，2005，24(4)：16-18
3廖洪利，王伟新，赵福胜，等．知母化学成分研究进展．药学实践杂志，2005，23(1)：12-14
4邓家刚，杨柯，阎莉，等．芒果苷对免疫抑制小鼠T淋巴细胞增殖的影响．中药药理与临床,2007，23(5)：64-65
5秦怀洲，王木梁，赵振伟，等．芒果苷对小鼠免疫功能影响的初步研究．中国临床药理学与治疗学,2007，12(8)：931-934
6农少云，农朝赞，潘莉莉，等．芒果苷对连坏蛋白P120磷酸化及肝癌细胞生物学行为的影响．广西医科大学学报,2005，22(4)：495-497
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12李瑞，孟庆刚.类风湿性关节炎的临床治疗研究．中华中医药学刊，2007，25（7）：1348-1350
13张相宏.类风湿性关节炎的中医发病机制探讨．甘肃中医，2007，20（4）：10-12
14王慧莲，周学平.系统性红斑狼疮的中医药研究进展．浙江中医药大学学报，2007，31（5）：792-794
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20王殿华，许凤全，张志慧.中医药治疗运动神经元病近况．山东中医杂志，2003,22（4）：252-254
21段秀梅，单俊杰，王喆星.皂苷治疗糖尿病及并发症的研究进展．中药材，2007，30（6）：748-750
22方朝晖，章小平.中医药改善2型糖尿病胰岛素抵抗研究思路与方法．中国中医药信息杂志，2003，10（12）：71-73
23谢纬，李丹，俞冰，张剑勇.中医药治疗痛风的进展．河南中医学院学报，2003，18（3）：86-88
24汤夏珍.内外兼治痛风性关节炎42例临床观察．浙江中医药大学学报，2007，31（6）：739-740
25王晓强.清热凉血法治疗初发Graves甲亢的理论探讨与临床研究．山东中医药大学，2007
26沈鸿，黄芳，窦昌贵.酸枣仁汤处方的配比研究．中国药科大学学报，2002，33（1）：21-23
27杨素德.中药药对知母—石膏配伍的化学成分变化及药效学研究．山东中医药大学，2012
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3Sun Y, Du Y, Liu Y, Chang L, Ren Y, Cao L, Sun Q, Shi X, Wang Q andZhang L. Simultaneous determination of9components in Anemarrhenaasphodeloides by liquid chromatography-tandem mass spectrometrycombined with chemometric techniques. Journal of separation science2012，35:1796-1807
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3DING J B, LI Y P, ZHANG J M, et al. Assay of sarsasapogenin inJinlianqingre Capsules by thin layer scanning chromatography. NingxiaMed J,2003，25（5）：274-276
4XU X J, XIE B X, LI Z M. Determination of indirubin in Jinlianqingreeffervescent tablets by HPLC. Strait Pharmaceutical Journal,2011，23（2）：39-40
5LIN Q F, FENG S Q, LI Y L, et al. Study on the antibacterial and antiviralactivity compositions of Trollius chinensis Bunge. Journal of ZhejiangUniversity(Science Edition),2004，31（4）：412-415
6Y.G. Sun, Y.F. Du, Y. Liu, L. Chang, Y.P. Ren, L. Cao, Q. Sun, X.W. Shi, Q.Wang, L.T. Zhang, Simultaneous determination of9components inAnemarrhena asphodeloides by liquid chromatography-tandem massspectrometry combined with chemometric techniques, J Sep Sci.35(2012)1796-1807
1国家药典委员会．中华人民共和国药典:2010年版一部［S］．北京:中国医药科技出版社，2010:820-821
2李明权.知柏地黄丸源流考.中医研究，1997，10(5)：56-57
3陈岳蓉，刘海青.高效液相色谱法测定知柏地黄丸中芒果苷的含量.中国医院药学杂志，2008，28（8）：677-679
4陈骁勇.HPLC法测定知柏地黄丸中菝葜皂苷元的含量.中国药房，2011，22（40）：3826-3827
5刘义梅.薄层色谱法测定知柏地黄丸中丹皮酚的含量.湖北中医杂志，2005，27（7）：52-54
6徐桂香，杨婕，陈钟文.HPLC法测定知柏地黄丸浓缩丸中马钱苷的含量.河南中医学院学报，2007，22（4）：30
7洪菁.HPLC法测定知柏地黄丸浓缩丸中丹皮酚的含量.海峡药学，2008，20（8）：31-33
8陈庆辉，唐元军.知柏地黄丸中盐酸小檗碱和盐酸巴马汀的含量测定.药物分析杂志，2012，32(12)：2198-2202
9翁晓明，李智林.高效液相色谱法测定知柏地黄丸中盐酸小檗碱和丹皮酚含量.中国医院药学杂志2001,21(1)：21-22
10Y.G. Sun, Y.F. Du, Y. Liu, L. Chang, Y.P. Ren, L. Cao, Q. Sun, X.W. Shi, Q.Wang, L.T. Zhang, Simultaneous determination of9components inAnemarrhena asphodeloides by liquid chromatography-tandem massspectrometry combined with chemometric techniques, J Sep Sci.2012，35：1796-1807
11易博，孙赫，原源，等．RP-HPLC法测定知母黄柏药对中新芒果苷、芒果苷和盐酸小檗碱．中草药，2007，38(6):856-858
12劳凤云,李小娜,张卫国,等．山茱萸总苷中莫诺苷及马钱苷HPLC法同时测定．中国公共卫生，2009，25（7）：889-891
13杨正生，彭振辉，姚青海，等．丹皮酚的药理作用研究进展．中国药物与临床，2011，11(5):545-547
1国家药典委员会．中华人民共和国药典（一部）[S].2010年版.北京：中国医药科技出版社，2010：194
2倪梁红，秦民坚．知母资源化学及药理研究进展．中国野生植物资源，2005，24(4)：16-18
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