一测多评法同时测定制川乌中9种生物碱的含量

目的:建立一测多评法同时测定制川乌中9种乌头类生物碱含量.方法:采用高效液相色谱四级杆飞行时间串联质谱(HPLC-QTOF-MS)法.色谱柱为Agilent ZORBAX Extend-C18 RRHT(2.1 mm×50 mm,1.8μm);甲醇为流动相A,以水(含0.1%甲酸和2.5 mmol·L-1醋酸铵溶液)为流动相B,梯度洗脱;流速为0.21 mL·min-1;柱温为30℃.以乌头碱为内参物,测定次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱、乌头原碱、次乌头原碱、新乌头原碱对于其的相对校正因子,并对相对校正因子进行重现性考察.利用相对校正因子计算其它8种生物碱成分的含量,同时利用外标法测定这9种成分,比较两种测定方法的差异,验证一测多评法的准确性和可行性.结果:建立QAMS法测定乌头类中药材9种生物碱的含量,其测定值与计算值无显著差异.结论:经方法学验证,本方法可用于制川乌药材中9种生物碱的含量测定.     

一测多评法测定小活络丸中9种乌头类生物碱的含量

摘 要 目的：建立一测多评法同时测定小活络丸中9种乌头类生物碱含量。方法: 采用高效液相色谱四级杆飞行时间串联质谱（HPLC QTOF MS）法,采用Agilent ZORBAX Extend C18 RRHT（2.1 mm×50 mm,1.8 μm）色谱柱;流速为0.21 ml·min-1;柱温为30 ℃;以甲醇为流动相A,以水（含0.1%甲酸和2.5 mmol·L^-1醋酸铵溶液）为流动相B,梯度洗脱。以乌头碱为内参物,测定次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱、乌头原碱、次乌头原碱、新乌头原碱对于其的相对校正因子,并对相对校正因子进行重现性考察。利用相对校正因子计算其他8种生物碱成分的含量,同时利用外标法测定这9种成分,比较两种测定方法的差异,验证一测多评法的准确性和可行性。结果: 在线性范围内,次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头碱、苯甲酰新乌头原碱、乌头原碱、次乌头原碱、新乌头原碱对乌头碱的相对校正因于分别为1.736、1.979、1.0471、0.9242、1.2901、1.3078、1.2859、1.0948,建立了QAMS法测定小活络丸中9种生物碱的含量,其测定值与计算值无显著差异。结论: 本文方法可用于小活络丸中乌头碱、次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱、乌头原碱、次乌头原碱、新乌头原碱的含量测定。     

乌头碱类成分中药制剂质量控制探讨

目的:探讨乌头碱类成分制剂的质量控制.方法:比较2005年版,2010年版中国药典对乌头类生物碱的质量控制要求,分析乌头碱类生物碱的毒性及其化学结构特征.结果与结论:2010年版药典与2005年版药典比较.2010年版药典对乌头类药材质量控制要求有利于药材使用的安全有效:2010年版药典对含乌头类生物碱成分的中药制剂的质量控制有待进一步加强.建议对含乌头类生物碱成分的中药制剂,在质量标准中研究建立双酯型生物碱的限量检查方法,以及双酯型生物碱水解产物苯甲酰新乌头原碱、苯甲酰乌头原碱和苯甲酰次乌头原碱等单酯型生物碱的总量测定,制定合理的含量范围,以保证用药的安全有效.     

佳蓉片中6种乌头类生物碱的定量研究

目的分析佳蓉片中苯甲酰新乌头原碱、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6种乌头类生物碱的含量。方法采用HPLC法。色谱条件:Kromasil C 18色谱柱(250 mm×4.6 mm,5μm);流动相为乙腈-四氢呋喃(25∶15)以及0.1 mol·L^-1醋酸铵溶液(每1000 mL加冰醋酸0.5 mL),梯度洗脱;流速为0.8 mL·min^-1;柱温为25℃;检测波长为235 nm。结果苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、乌头碱和次乌头碱分别在4.32~432.40,1.29~128.83,1.08~108.46,1.77~177.30,0.78~78.40和1.19~119.04μg·mL^-1范围内与峰面积线性关系良好;平均回收率分别为96.46%,98.26%,98.03%,98.23%,98.86%和98.70%;RSD值分别为1.50%,1.20%,1.27%,1.06%,1.50%和1.19%;10批制剂中苯甲酰新乌头原碱、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含量范围分别为100.32~119.18,9.35~16.02,7.99~10.87,1.54~1.85,0.87~0.99和2.21~3.27μg·g^-1。结论该方法简单、准确、灵敏、重复性好,可作为佳蓉片中乌头类生物碱成分的定量方法。     

附子总生物碱提取物中3个双酯型和3个单酯型乌头碱成分的含量测定

目的 提取附子总生物碱,采用高效液相色谱法同时测定附子总生物碱提取物中苯甲酰新乌头碱、苯甲酰乌头原碱、苯甲酰乌头次碱、新乌头碱、乌头碱和次乌头碱6个成分的含量.方法 采用85％的乙醇回流提取附子药材,经大孔吸附树脂柱分离后,制得附子总生物碱提取物,采用Waters Terra C18色谱柱(3.0 mm×100mm,3.5μm);流动相为A:乙腈,B:水(5 mM醋酸铵),梯度洗脱,A相随时间的变化:15％ ～26％ (0～ 10 min),26％ ～30％(10 ～ 20min),30％ (20 ～25 min);流速0.5 ml/min;检测波长235 nm;柱温25℃;进样量5μl.结果 6个生物碱成分苯甲酰新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头碱、新乌头碱、乌头碱、次乌头碱在20 min内实现完全分离,在11.40～114.0、10.22 ～ 102.2、10.36 ～ 103.6、6.250 ～62.50、13.50 ～135.0、15.12～151.2 μg/ml的线性范围内,线性关系良好(r ＞0.999).方法学考察结果表明,日内日间精密度,最低检测限和定量限的范围均符合含量测定的要求,加样回收率(n=6)分别为104.52％ (RSD=2.0％)、103.43％(RSD=1.6％)、101.92％ (RSD =2.3％)、99.45％ (RSD =2.7％)、97.67％(RSD=1.9％)、101.15％(RSD=3.4％).结论 该方法简便、准确、可靠,可用于附子提取物中6个乌头碱成分的含量测定.     

虎力散片中乌头类生物碱成分HPLC特征图谱及多成分含量测定

目的 建立虎力散片中乌头类生物碱的HPLC特征图谱，并同时测定6种生物碱含量。方法 采用Kromasil 100-5-C₁₈色谱柱（4.6 mm×250 mm，5 μm），以乙腈（A）-0.2%乙酸溶液（三乙胺调pH至6.20）（B）为流动相，梯度洗脱，流速1.0 mL·min^–1，柱温35℃，检测波长235 nm。采用中药色谱指纹图谱相似度评价系统对结果进行分析，并结合聚类分析、主成分分析等模式识别技术对样品的总体质量进行分析评价，并对主要有效成分进行定量测定。结果 11批虎力散片的乌头类生物碱特征图谱中共确认10个共有峰，与混合对照品比较确定6个生物碱：苯甲酰新乌头原碱、苯甲酰次乌头原碱、苯甲酰乌头原碱、新乌头碱、次乌头碱、乌头碱。来自3个厂家的11批虎力散片中苯甲酰新乌头原碱、苯甲酰次乌头原碱、苯甲酰乌头原碱、新乌头碱、次乌头碱、乌头碱的含量分别在231.190~1 002.977，17.022~105.744，21.962~48.406，5.436~39.678，2.692~97.854，1.707~43.342 μg·g^–1。结论 所建立的HPLC特征图谱方法和多成分含量测定方法，简便可行，结果准确可靠，重现性好，可有效评价虎力散片的质量。     

三七伤药片中6种乌头类生物碱含量测定及化学计量学分析

目的：采用高效液相色谱法，建立三七伤药片中乌头碱、次乌头碱、新乌头碱与苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱6种生物碱类成分的含量测定方法，并根据测定结果进行化学计量学分析。方法：采用混合型阳离子交换反相吸附树脂(MCX)固相萃取小柱对供试品进行纯化，高效液相色谱法测定，使用COSMOSIL C₁₈色谱柱(4.6 mm×250 mm,5μm)，以甲醇-乙腈-0.1%磷酸为流动相，梯度洗脱，流速为1.0 mL·min^-1，柱温为25℃，检测波长为232 nm。采用SIMCA软件对结果进行处理，运用主成分分析法分析影响产品差异的关键因素。结果：乌头碱、次乌头碱、新乌头碱与苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱分别在各自范围内线性关系良好(r=0.9999)，平均加样回收率(RSD)分别为101.8%(1.6%)、100.6%(1.4%)、97.8%(1.1%)、100.8%(1.5%)、101.0%(1.6%)、100.7%(0.8%)。17批次样品测定结果显示不同批次之间6种生物碱含量存在较大差异；单酯型生物碱的含量是影...     

一测多评法测定黄连须中的5种生物碱

目的 采用一测多评法同时测定黄连须中5种生物碱(盐酸小檗碱、盐酸药根碱、盐酸表小檗碱、盐酸黄连碱和盐酸巴马汀)的含量.方法 以盐酸小檗碱为内参物,采用HPLC法测定其与盐酸药根碱、盐酸表小檗碱、盐酸黄连碱、盐酸巴马汀的相对校正因子,利用该相对校正因子计算其他4种生物碱的含量;同时利用外标法测定5种生物碱的含量,比较两种测定方法的差异,验证一测多评法的可行性和准确性.结果 在线性范围内,盐酸小檗碱与盐酸药根碱、盐酸表小檗碱、盐酸黄连碱、盐酸巴马汀的相对校正因子分别为1.128、1.008、1.070、1.025,在不同实验条件下,相对校正因子的重复性良好,5种生物碱成分含量的计算值与实测值间无显著性差异.结论 一测多评法同时测定黄连须中5种生物碱的含量是可行的、准确的.     

一测多评法测定华佗豆4种生物碱

目的 建立一测多评法检测华佗豆药材中4种生物碱的量。方法 以华佗豆碱乙为内标,建立华佗豆碱甲、碱丙、碱丁的相对校正因子,在考察相对校正因子的重现性后,进一步采用外标法与一测多评法所计算出的成分含量进行比较。结果 6批华佗豆药材中,各成分采用相对因子计算的量与采用外标法计算的结果无显著差异。结论 一测多评法操作简单、结果准确、可靠,可用于华佗豆4种生物碱的测定。     

固相萃取辅助UPLC-MS/MS同时测定肾康宁胶囊中乌头类生物碱的含量

目的:建立肾康宁胶囊中乌头类生物碱的含量测定方法。方法:采用Waters ACQUITY UPLC HSS T3色谱柱(2.1 mm×100 mm, 1.8μm),以0.1%甲酸水溶液(A)-乙腈(B)为流动相,梯度洗脱,流速0.3 mL·min^-1,柱温40℃;质谱采用电喷雾离子化源(ESI),正离子模式下选择多反应监测(MRM)模式检测,建立同时测定肾康宁胶囊中乌头碱、新乌头碱、次乌头碱、苯甲酰乌头原碱、苯甲酰新乌头原碱、苯甲酰次乌头原碱、乌头原碱和新乌头原碱8个生物碱含量的固相萃取(SPE)辅助UPLC-MS/MS分析方法。结果:乌头碱、新乌头碱、次乌头碱、苯甲酰乌头原碱、苯甲酰新乌头原碱、苯甲酰次乌头原碱、乌头原碱、新乌头原碱分别在0.31～99.80、0.78～99.80、0.31～75.15、30.31～485.00、309.38～3 712.50、61.88～742.50、7.75～248.00、61.25～1 960.00 ng·mL^-1的质量浓度范围内线性关系良好,精密度、重复性和稳定性的RSD均小于5%,加样回收率在80....     

Separation and identification of Aconitum alkaloids and their metabolites in human urine.

To study the safety of Aconitum medicinal herbs in clinic and identify Aconitum alkaloids poisoning in forensic medicine, Aconitum alkaloids and their metabolites were separated and identified in human urine by liquid chromatography-electrospray ionization-multi-stage mass spectrometry (LC-ESI-MS(n)) and chemical pathway of metabolism was investigated. The alkaloids and their metabolites in the urine sample were extracted with solid-phase cartridges and separated by HPLC with acetonitrile-water-formic acid (40:60:0.5) mobile phase. Structures of five metabolites and three parent Aconitum alkaloids were identified with multi-stage mass spectrometry data through comparison with authentic substances as aconitine (M(1)), mesaconitine (M(2)), hypaconitine (M(3)), benzoylaconine (M(4)), benzoylmesaconine (M(5)), benzoylhypaconine (M(6)), 16-O-demethylaconitine (M(7)) and 16-O-demethylhypaconitine (M(8)), respectively. Among them, M(8) was identified and reported for the first time. Metabolic pathways of Aconitum alkaloids in human body were proposed.     

Quantitative determination of diterpenoid alkaloids in four species of Aconitum by HPLC

A high performance liquid chromatography (HPLC) method has been developed for the determination of five principal alkaloids (benzoylmesaconine, mesaconitine, aconitine, hypaconitine and deoxyaconitine) found in four species of genus Aconitum. The five alkaloids were analyzed simultaneously with an XTerraRP18 column by gradient elution using 0.03 M ammonium hydrogen carbonate-acetonitrile as mobile phase. The recovery of the method was 94.6-101.9%, and all the alkaloids showed good linearity (gamma = 0.9999) in a relatively wide concentration range. The results indicated that contents of alkaloids in Aconitum varied significantly from species to species; hence quality control of Aconitum drugs is very necessary.     

Development and validation of a highly sensitive UPLC-MS/MS method for simultaneous determination of aconitine, mesaconitine, hypaconitine, and five of their metabolites in rat blood and its application to a pharmacokinetics study of aconitine, mesaconitine, and hypaconitine

A rapid, specific and sensitive method was developed for the simultaneous determination of eight Aconitum alkaloids: aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine and mesaconine in rat blood by ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS). The UPLC-MS/MS system coupled with an electrospray ionization (ESI) source was operated in a positive mode via multiple-reaction monitoring (MRM). Samples were treated with methanol to remove protein prior to analysis by UPLC-MS/MS. The analytes were separated with a Waters C18 column (1.7 μm, 50?×?2.1?mm) and a gradient elution using acetonitrile and 0.1% formic acid-water as the mobile phases. The linear response range was from 0.125 to 1000 nmol/L for these eight alkaloids and the correlation coefficients (r(2) values) were all higher than 0.997. The method was validated with respect to precision, accuracy, recovery, matrix effect, carryover effect and sample stability, and found to be within the acceptable limits. The developed and validated method was successfully applied to simultaneously determine the eight Aconitum alkaloids in rats blood after intravenous administration of a mixture of AC, MA and HA.     

反相离子对色谱法测定附子中生物碱成分

目的测定附子中乌头碱、新乌头碱、次乌头碱、北乌碱、苯甲酰乌头原碱和苯甲酰新乌头原碱等6种生物碱的含量。方法用反相离子对HPLC法，使用AichromBond-1 C₁₈柱(250 mm×4.6 mm ID)；流动相：乙腈-5 mmol·L^-1 NaH₂PO₄溶液，磷酸调至pH 4.5(50∶50)，内含7 mmol·L^-1 十二烷基硫酸钠(SDS)；检测波长：235 nm；流速：1.0 mL·min^-1；柱温：35 ℃。结果以上6种生物碱可以完全分离，准确测定。结论该方法准确度高，可应用于附子中生物碱的含量测定。     

附子提取液的稳定性研究

目的研究附子提取液加热过程中酯型生物碱含量的动态变化,并考察其稳定性及其变化规律。方法采用RP-HPLC法测定提取液中酯型生物碱的含量。色谱柱为Eclipse XDB-C18柱(250 mm×4.6 mm,5μm),流动相为0.1 mol.L-1乙酸铵溶液(含0.05%冰乙酸)为A相,乙腈-四氢呋喃(25∶15)为B相,梯度洗脱,检测波长235 nm。结果附子提取液(pH5.9)于95℃加热10 h,苯甲酰新乌头原碱和苯甲酰次乌头原碱的含量先增后降,苯甲酰乌头原碱的含量缓慢降低,而新乌头碱、次乌头碱含量迅速降低;附子提取液(pH3)在相同加热条件下,苯甲酰新乌头原碱和苯甲酰次乌头原碱的含量缓慢增加,苯甲酰乌头原碱、新乌头碱、次乌头碱含量的缓慢降低,但总体较稳定;附子提取液为pH9时,酯型生物碱的含量均迅速下降。结论 pH、加热时间对附子提取液中酯型生物碱的稳定性有显著影响。     

全固态乌头碱电化学检测器的研制及其在流动注射分析中的应用

报道一种新型结构的全固态乌头碱电化学检测器的研制及其在流动注射分析中的应用。采用流动注射分析法对川乌、草乌及其中成药(小活络丸)中剧毒成分(双酯型生物碱)进行了测试,方法简便快速,结果同光度法接近。本文还提出了电化学法研究乌头碱水解动力学原理。在pH 6.5,温度98℃的条件下测得乌头碱水解成苯甲酰乌头碱的速度常数为1.36×10^-2min^-1。     

Physicochemical quality evaluation of natural compounds isolated from crude drugs

Aconite root has high toxicity caused by diester alkaloids, thus it was necessary to define the limiting value of diester alkaloids used in medicine formulation. To give the quality of “Processed Aconite Root” and “Powdered Processed Aconite Root” in the Japanese Pharmacopoeia (14th edn, supplement II), we established the official specification and evaluation methods of standard substances. High qualitative grade diester alkaloids, aconitine, hypaconitine, jesaconitine and mesaconitine, which were useful to evaluate the purity of processed aconite root and powdered processed aconite root, were prepared and evaluated for their stability. We studied the physicochemical specification and evaluation methods of these alkaloids. In addition, an “Aconitum diester alkaloids standard solution for purity”, which was used for the purity test, was prepared, and we also studied its physicochemical specification and evaluation methods. In addition, to evaluate the quality of processed aconite root and powdered processed aconite root, a TLC identification test was established. A monoester alkaloid of benzoylmesaconine hydrochloride was used as the reference standard in the latter test, and we also investigated its physicochemical specification and evaluation methods.     

Determination of Aconitum alkaloids in the tubers of Aconitum japonicum using gas chromatography/selected ion monitoring

A rapid, specific and precise method using GC/SIM was applied to separate and quantify Aconitum alkaloids in Aconitum tubers. The TMS derivatives of each alkaloid produced a well defined peak on selected ion recording (SIR). Good linear response over the range of 100 pg - 7.5 ng was demonstrated for each alkaloid. Furthermore, we investigated the changes in the contents of Aconitum alkaloids, i.e., hypaconitine, mesaconitine, aconitine, and jesaconitine, in Aconitum tubers paying special attention to the harvesting season. Mesaconitine had the highest value in all seasons. Mesaconitine and aconitine showed the highest value in the samples harvested in May 1991 while hypaconitine had the highest value among those harvested in December 1990. As to the total amount of the alkaloids, the highest value, 4190 microg/g, and the lowest value, 1881 microg/g, were observed in the samples harvested in May and September, respectively. GC/SIM was very useful for the determination of Aconitum alkaloids in the tubers.