Temperature and Acidified Solvent Effect on Total Anthocyanins and RP-HPLC Phenolic Acids Determination in Selected Spices

Total anthocyanins of spices (Syzygium aromaticum L., Coriadrum sativum L., Cuminum cyminum L., Zingiber officinale Rosc., Elettaria cardamomum, Curcuma longa, Rhus coriaria L., Cinnamomum zeylanicum Blume, Foeniculum vulgare Mill and Laurus nobilis L.) were determined using acidified (1% HCl) solvents (methanol, ethanol and acetone) at three temperatures (20℃, 40℃ and 60℃). Also phenolic acids were separated and identified by RP-HPLC. Results showed that sumac and cinnamon had the highest levels of anthocyanins, while for the acetone the cinnamon indicated the highest amount of anthocyanins when methanol and ethanol were used as extracting solvents at 20℃. At 40℃ using ethanol, sumac showed the highest level of anthocyanins whereas acetone solvent yielded the highest anthocyanin contents for cinnamon. At 60℃, cinnamon showed the highest level of anthocyanins when methanol and acetone were the solvents, while sumac had the highest anthocyanins level using ethanol as solvent. HPLC results showed ten phenolic acids found in those spices and varied in their concentrations. Gallic acid had the highest level (1642.3 mg/100g) (cloves). Gentisic acid had the lowest level (1.2 mg/100g) in ginger. Also sumac showed the highest level of chlorogenic acid (1528.7 mg/100g). Some acids were not found in some spices, for instance, benzoic acid was not found in coriander, cumin, ginger, green cardamom, cinnamon and sweet laurel.

Application of HPLC and ESI-MS techniques in the analysis of phenolic acids and flavonoids from green leafy vegetables (GLVs)

Diets containing high proportions of fruits and vegetables reduce the risk of onset of chronic diseases. The role of herbal medicines in improving human health is gaining popularity over the years, which also increases the need for safety and efficiency of these products. Green leafy vegetables （GLVs） are the richest source of phenolic compounds with excellent antioxidant properties. Increased consumption of diets containing phenolic compounds may give positive and better results to human health and significantly improves the immune system. Highly selective, susceptible and versatile analytical techniques are necessary for extraction, identifica- tion, and quantification of phenolic compounds from plant extracts, which helps to utilize their important biological properties. Recent advances in the pre-treatment procedures, separation techniques and spectro- metry methods are used for qualitative and quantitative analysis of phenolic compounds. The online coupling of liquid chromatography with mass spectrometry （LC-MS） has become a useful tool in the metabolic profiling of plant samples. In this review, the separation and identification of phenolic acids and flavonoids from GLVs by LC-MS have been discussed along with the general extraction procedures and other sources of mass spectrometer used. The review is devoted to the understanding of the structural configuration, nature and accumulation pattern of phenolic acids and flavonoids in plants and to highlighting the recent developments in the chemical investigation of these compounds by chromatographic and spectroscopic techniques. It concludes with the advantages of the combination of these two methods and prospects.

Identification and characterization of phenolics and terpenoids from ethanolic extracts of Phyllanthus species by HPLC-ESI-QTOF-MS/MS

Phyllanthus species plants are a rich source of phenolics and widely used due to their medicinal properties. A liquid chromatography–tandem mass spectrometry(LC–MS/MS) method was developed using high-pressure liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(HPLC-ESI-QTOFMS/MS) for the identification and characterization of quercetin, kaempferol, ellagic acid and their derivatives in ethanolic extracts of Phyllanthus species. The chromatographic separation was carried out on Thermo Betasil C_8 column(250 mm×4.5 mm, 5 μm) using 0.1% formic acid in water and 0.1% formic acid in methanol as the mobile phase. The identification of diagnostic fragment ions and optimization of collision energies were carried out using 21 reference standards. Totally 51 compounds were identified which include 21 compounds identified and characterized unambiguously by comparison with their authentic standards and the remaining 30 were tentatively identified and characterized in ethanolic extracts of P. emblica, P. fraternus, P. amarus and P.niruri.

PHENOLIC ANTIOXIDANTS DETERMINATION IN FOOD ITEMS USING REVERSED-PHASE HPLC

The determination of synthetic phenolic antioxidants （SPAs） including propyl gallate （PG）, tertiary butyl hydroquinone （TI3HQ）, butylated hydroxyanisole （BHA） and butylated hydroxytoluene （BHT） in food items is reported using high performance liquid chromatography （HPLC）. A Cls column is used as the stationary phase, acetonltrile and water：Acetic acid （1%） is used as the mobile phase of gradient elution and the UV detec- tor is set at 280 nm. Under the above conditions, four antioxidents is completely separated within 8 rain. The limit of detection, linear range, and reproducibility of HPLC are evaluated. Isolation parameters of SPAs from different types of food items （cooking oil, margarine and butter, and cheese） are optimized. SPAs are extracted from food items through extraction with methanol/acetonitrile （1 ： 1, in volume）, vortex, ultrasonic treatment and precipitation in a freezer （2 h）. For cooking oil margarine, butter and cheese at 50 and 200 rag/L, recoveries of SPAs are 93.3%0--108.3% （PG）, 85.3~^--108.3~~ （TBHQ）, 96.7~^--101.2~/6 （BHA）, and 73.9^-- 94.6% （BHT）. The method is applied to the determination of SPAs in 38 food items （16 cooking oils, 8 mar- garine, 6 butter and 6 cheese samples）. The levels of SPAs in positive samples are all below the legal limits of China.

HPLC法同时测定脑脉泰胶囊中12种成分的含量

目的建立HPLC法同时测定脑脉泰胶囊中3′-羟基葛根素、2-羟基红花黄色素A、葛根素、葛根素芹菜糖苷、3′-甲氧基葛根素、大豆苷、二苯乙烯苷、木犀草苷、染料木苷、木犀草素、丹酚酸B、迷迭香酸的含量。方法分析采用KromasilC_(18)色谱柱(250mm×4.6mm,5μm);流动相乙腈-水(含1.0g/L磷酸),梯度洗脱;体积流量0.8mL/min;柱温30℃;检测波长280nm。再进行聚类分析、主成分分析。结果12种成分在各自范围内线性关系良好(r>0.9990),平均加样回收率97.07%~98.30%,RSD0.86%~1.82%。10批样品聚为4类,4种主成分累积方差贡献率达86.262%。结论该方法简便稳定,可靠准确,可为脑脉泰胶囊的质量控制提供科学依据。

引火汤冻干粉HPLC指纹图谱建立及8种成分含量测定

目的建立引火汤冻干粉HPLC指纹图谱,并测定地黄苷D、麦冬甲基黄烷酮A、毛蕊花糖苷、原儿茶酸、梓醇、五味子醇甲、五味子乙素、五味子甲素的含量。方法指纹图谱建立采用Supersil AQ18色谱柱(4.6 mm×250 mm,5μm);流动相乙腈-0.1%磷酸,梯度洗脱;体积流量1.0 mL/min;柱温30℃;检测波长230 nm。UPLC-MS/MS含量测定采用Alphasil VC-C_(18)色谱柱(2.1 mm×100 mm,2.5μm);流动相乙腈-0.1%甲酸,梯度洗脱;体积流量0.3 mL/min;柱温30℃;电喷雾离子源;正负离子扫描;多反应监测模式。结果10批冻干粉指纹图谱中有17个共有峰,相似度大于0.90,指认出毛蕊花糖苷、水晶兰苷、去乙酰车叶草苷酸、五味子醇甲。8种成分在各自范围内线性关系良好(R^(2)>0.9906),平均加样回收率98.7%~101.1%,RSD 2.0%~5.2%。结论指纹图谱结合含量测定能完整表征引火汤基准样品质量,从而为其关键化学属性质量评价提供参考。

HPLC-UV法同时测定赤血康脉胶囊中五种成分

本研究旨在建立HPLC-UV法同时测定赤血康脉胶囊中芍药苷、毛蕊异黄酮葡萄糖苷、β-蜕皮甾酮、阿魏酸和党参炔苷的含量。采用DiamonsilR C_(18)(250 mm×4.6 mm,5μm)色谱柱;流动相为乙腈-0.1%磷酸(梯度洗脱);流速为1.0 mL/min;柱温为35℃;检测波长为268 nm,进行分析。结果显示,5种化学成分在各自范围内线性关系良好(r>0.9995);精密度、稳定性、重复性试验结果的RSD均小于2.0%,平均加样回收率99.35%~101.59%,RSD 0.95%~2.69%。该含量测定方法简单可靠、重复性好,操作便捷,适用于同时测定赤血康脉胶囊中五种成分的含量。

HPLC-ELSD同时测定腺梗豨莶草中6个二萜成分的含量

为了研究腺梗豨莶草中6个二萜成分(对映海松烯型二萜:奇任醇、Hythiemoside B和豨莶精醇;对映贝壳杉烷型二萜:对映-17,18-二羟基-贝壳杉烷-19-羧酸、对映-16β,17-二羟基-贝壳杉烷-19-羧酸和对映-16α氢-贝壳杉烷-17,19-二羧酸)的含量,本试验建立了高效液相色谱法-蒸发光散射检测器(HPLC-ELSD)同时测定二萜类化合物含量。样品粉碎过筛加甲醇和乙酸乙酯回流提取,蒸发减压除去溶剂,甲醇溶解,0.45μm滤膜滤过,取续滤液进行测定。色谱条件:色谱柱为Waters Symmetry Shield^(TM)RP18柱(250 mm×4.6 mm,5μm),流动相为0.3%甲酸水溶液-乙腈(v/v),梯度洗脱,流速为1.0 mL/min。蒸发光散射检测器漂移管温度为103℃,雾化气流速为3.0 L/min。应用该方法测定腺梗豨莶草样品不同部位中6个二萜类化合物的含量,同时比较叶、枝、茎中对映海松烯型二萜、对映贝壳杉型二萜和总二萜含量的差异。结果显示,6个二萜成分在其线性范围内线性关系良好(r≥0.9992);日内和日间精密度相对标准偏差(RSD)均小于3.5%;回收率介于96.5%~101.5%,RSD均小于2.3%;腺梗豨莶草不同部位(叶、枝、茎)的二萜类化合物含量差异较大。结果表明,本试验所建立的HPLC-ELSD方法简便、准确、重复性好,为腺梗豨莶草药材全面的质量评价和临床应用中最佳药用部位的选择提供了参考。

基于HPLC-QQQ-MS技术探讨炮制对肉苁蓉中5个活性成分的含量影响

炮制一般会对中药材的成分及药效产生重要作用,本文为了探讨炮制加工对肉苁蓉成分的影响,建立了同时测定炮制前后肉苁蓉中肉苁蓉苷F、毛蕊花糖苷、毛蕊花糖苷、2′-乙酰基毛蕊花糖苷和松果菊苷的含量的高效液相色谱-串联三重四极杆质谱法(high-performance liquid chromatography-tandem triple quadrupole mass spectrometry,HPLC-QQQ-MS)的检测方法。采用50%甲醇溶液提取药物粉末,在Agilent Zorbax Eclipse Plus C 18(2.1 mm×150 mm,1.8μm)的色谱柱上,以0.1%甲酸水(A)-乙腈(B)为流动相进行梯度洗脱,选取负离子多反应监测(multiple reaction monitoring,MRM)模式,以木通苯乙醇苷为内标成分,以进样量1μL,流速0.3 mL/min,柱温35℃,样品管理器温度8℃于HPLC-QQQ-MS进行同一批次炮制前后肉苁蓉中这五种成分的含量测定。结果显示5种成分在各自范围内线性关系良好,相关系数(r)在0.9929~0.9985之间,平均加样回收率在98.15%~100.2%,相对标准偏差(relative standard deviation,RSD)小于4.6%。所建立的研究方法简便准确,可用于肉苁蓉的质量控制,并用于评价炮制对肉苁蓉含量及药效的影响。

HPLC法同时测定补肾活血散结胶囊中12种成分的含量

目的建立HPLC法同时测定补肾活血散结胶囊中没食子酸、原儿茶酸、莫诺苷、马钱苷、獐牙菜苷、芍药苷、金丝桃苷、紫云英苷、丹酚酸B、丹酚酸A、朝藿定C、淫羊藿苷的含量。方法分析采用Agilent 5 TC-C_(18)色谱柱(250 mm×4.6 mm,5μm);流动相乙腈-0.1%磷酸,梯度洗脱;体积流量1.0 mL/min;柱温30℃;检测波长240 nm。结果12种成分在各自范围内线性关系良好(r≥0.9998),平均加样回收率97.11%~101.14%,RSD 0.60%~2.65%。结论该方法简便准确,重复性好,可用于补肾活血散结胶囊的质量控制。

HPLC法同时测定3种滑膜炎制剂中9种成分的含量

目的 建立HPLC法同时测定滑膜炎颗粒、胶囊、片剂中迷迭香酸、丹酚酸B、丹酚酸A、丹参素、原儿茶醛、绿原酸、隐绿原酸、新绿原酸、咖啡酸的含量。方法 分析采用Xselect■HSS T3色谱柱(250 mm×4.6 mm, 5μm);流动相乙腈-0.1%磷酸,梯度洗脱;体积流量1 mL/min;柱温30℃;检测波长280、287、327 nm。结果 9种成分在各自范围内线性关系良好(r>0.999 0),平均加样回收率97.05%~102.14%,RSD 0.91%~2.59%。不同制剂中丹酚酸B含量均符合2020年版《中国药典》要求,各成分综合含量依次为颗粒>胶囊>片剂(B公司)>片剂(E公司)>片剂(D公司)>片剂(C公司)。结论 该方法简便、快速、稳定,结果准确可靠,可为滑膜炎制剂质量标准提升提供参考依据。

HPLC-UV法测定舒肝和胃丸中α-香附酮和香附烯酮的含量

目的:建立舒肝和胃丸中α-香附酮和香附烯酮的高效液相色谱(HPLC-UV)含量测定法,为该制剂中醋香附含量的质量控制提供依据。方法:对舒肝和胃丸中α-香附酮和香附烯酮同时进行含量测定。采用Agilent ZORBAX SB-C18色谱柱(4.6 mm×250 mm,5μm),流动相为甲醇和水(65∶35),流速1.0 mL·min^(-1),检测波长254 nm,柱温30℃,进样量5μL。结果:采用该方法得到的舒肝和胃丸色谱图中,α-香附酮和香附烯酮色谱峰与相邻色谱峰分离效果较好,满足含量测定的要求;指标性成分α-香附酮、香附烯酮分别在0.08036~0.80360μg、0.2112~2.1120μg范围内线性关系良好,相关系数均为1.000;平均加标回收率分别为102.0%(RSD:1.6%,n=6),102.4%(RSD:2.0%,n=6);稳定性(24 h)、重复性、精密度良好。结论:本研究建立的α-香附酮和香附烯酮HPLC-UV含量测定方法简单、快捷、准确,可为舒肝和胃丸中醋香附含量的控制提供依据。

葛藤花HPLC指纹图谱建立及3种成分含量测定

目的建立葛藤花HPLC指纹图谱,并测定染料木苷、鸢尾苷、染料木素的含量。方法分析采用AQ-C_(18)色谱柱(250 mm×4.6 mm,5μm);流动相乙腈-0.1%磷酸,梯度洗脱;体积流量1.0 mL/min;柱温30℃;检测波长260 nm。再进行聚类分析和主成分分析。结果10批药材指纹图谱中有17个共有峰,相似度大于0.970。3种成分在各自范围内线性关系良好(r>0.9990),平均加样回收率100.50%~104.30%,RSD 1.93%~5.21%。各批药材聚为2类,3种主成分累积方差贡献率为84.822%。结论该方法稳定可靠,可用于葛藤花的质量控制。

HPLC-DAD双波长法同时测定鼠尾草中3个酚酸类成分的含量

建立鼠尾草中咖啡酸、原儿茶酸和迷迭香酸3个酚酸类成分含量测定的方法。采用SHIMADZU VP-ODS色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.1%磷酸水(梯度洗脱),检测波长为260 nm和327 nm,柱温为30℃。结果表明咖啡酸、原儿茶酸、迷迭香酸的线性范围分别是0.45~22.53μg/mL(r=0.9999)、0.21~10.34μg/mL(r=0.9999)、0.53~21.19μg/mL(r=0.9999),平均加样回收率分别为98.60%、100.32%、99.28%,RSD分别为1.43%、1.90%、0.91%。本方法简单快速,重复性好,可用于鼠尾草中上述3种酚酸类成分的含量测定。

HPLC法同时测定知黄抗癌方中7种成分的含量

目的建立HPLC法同时测定知黄抗癌方中红景天苷、绿原酸、芒果苷、虎杖苷、盐酸小檗碱、异牡荆素、白藜芦醇的含量。方法分析采用SPOLARC_(18)色谱柱(4.6mm×250mm,5μm);流动相乙腈-0.1%磷酸,梯度洗脱;体积流量1.0mL/min;柱温30℃;检测波长275nm。结果7种成分在各自范围内线性关系良好(r≥0.9997),平均加样回收率91.32%~106.95%,RSD0.4%~2.9%。结论该方法简便快速,专属性好,可用于知黄抗癌方的质量控制。

鱼腥草芩蓝合剂HPLC指纹图谱及8种成分含量测定

目的:建立鱼腥草芩蓝合剂的HPLC指纹图谱,并同时测定其中黄芩苷、汉黄芩苷、汉黄芩素、连翘苷、连翘酯苷A、绿原酸、芦丁和槲皮苷的含量。方法:采用SHIMSEM Ankylo C_(18)色谱柱(4.6 mm×250 mm,5μm);流动相为甲醇-0.1%磷酸,梯度洗脱;检测波长210 nm;体积流量1.0 mL/min;柱温40℃;进样量10μL。以黄芩苷作为参照峰,建立17批鱼腥草芩蓝合剂的指纹图谱,综合应用相似度评价、聚类分析和主成分分析评价鱼腥草芩蓝合剂批间质量差异;并同时测定其中8种成分的含量。结果:17批样品中有27个共有峰,相似度均>0.990。聚类分析可将样品分为5类,筛选出3个主成分,累计贡献率75.804%。8种成分在各自范围内线性关系良好(r≥0.9995),平均加样回收率为96.17%~103.53%,RSD为0.50%~1.23%。结论:该试验方法稳定、可靠,可对鱼腥草芩蓝合剂进行质量控制。

虎克毛冠菊的化学成分研究及2种成分的HPLC法测定

目的系统提取、分离和纯化虎克毛冠菊中的化学成分,从中筛选出2种主要成分,作为质量标志物对15批虎克毛冠菊药材进行HPLC质量分数测定。方法采用HP20大孔树脂柱层析、半制备型高效液相色谱等多种分离方法对虎克毛冠菊乙醇提取物进行分离纯化,运用现代波谱方法(UV、IR、MS、NMR)鉴定化合物的结构。选取质量分数最高的2种主要成分鸢尾苷、鸢尾甲苷A(质量分数≥98%)作为对照品,采用Gemini-NX C18(250 mm×4.6 mm,5μm)色谱柱,以乙腈(A)-0.2%甲酸(B)溶液为流动相进行梯度洗脱;柱温为30℃;流速为1.0 mL/min;进样量为10μL;检测波长为265 nm。对15批虎克毛冠菊药材中鸢尾苷、鸢尾甲苷A的质量分数进行测定。结果从虎克毛冠菊中共分离得到8个化合物,分别鉴定为鸢尾甲苷A(1)、鸢尾苷(2)、鸢尾新苷B(3)、野鸢尾苷(4)、鸢尾苷元(5)、野鸢尾苷元(6)、毛蕊异黄酮(7)、鸢尾甲黄素B(8),其中鸢尾苷、鸢尾甲苷A的质量分数最高。15批虎克毛冠菊药材中鸢尾苷、鸢尾甲苷A质量分数分别为37.26~53.80 mg/g、29.39~39.44 mg/g。结论化合物1-8均为首次从该植物中分离得到,15批虎克毛冠菊药材的质量分数测定结果准确可靠。拟定本品按干燥品计算,鸢尾苷质量分数不低于3.8%,鸢尾甲苷A质量分数不低于3.0%,为虎克毛冠菊质量标准的建立提供了参考。

基于HPLC指纹图谱的高酚酸耐盐金银花品种筛选

为了筛选出高酚酸含量且耐盐的金银花品种,在河北、山东、河南不同地点采集金银花品种17个,统一移植到河北省农林科学院滨海农业研究所实验基地的盐渍土(NaCl含量0.1%)上栽培繁殖,盐胁迫14 d后取枝条进行酚酸物质含量检测。采用高效液相色谱法(HPLC)与指纹图谱综合质量评价相结合的方法,以酚酸含量高为目标,对金银花品种进行筛选。结果表明:不同的金银花品种具有共同的特征色谱峰12个,特征峰中以绿原酸、异绿原酸A、异绿原酸C、木犀草苷这4种酚酸物质含量表现较高;不同品种的酚酸物质含量差异较大,其中变异株的绿原酸(15.7 mg/g)以及总酚酸物质含量(20.35 mg/g)均为最高,其次是古郡红(绿叶)(绿原酸含量为11.87 mg/g,总酚酸物质含量为17.05 mg/g),二者指标值均显著>其他品种。变异株和古郡红(绿叶)为本次试验筛选出的适合在盐碱地推广种植的品种。

基于HPLC和化学计量法分析蜘蛛香不同部位3类化合物10种成分差异研究

目的 对蜘蛛香不同部位中酚酸类(新绿原酸、绿原酸、咖啡酸、异绿原酸B、异绿原酸A、异绿原酸C),黄酮类(橙皮苷、蒙花苷)及缬草三酯类(乙酰缬草三酯、缬草三酯)化合物含量进行测定,结合化学模式识别比较含量差异。方法 采用HPLC法对蜘蛛香地上部位(叶)和地下部位(根茎和根)中10种成分含量进行测定,对结果进行聚类分析、主成分分析和正交偏最小二乘判别分析。结果 地上部位10种成分平均含量绿原酸>异绿原酸A>蒙花苷>缬草三酯>橙皮苷>异绿原酸C>乙酰缬草三酯>异绿原酸B>新绿原酸>咖啡酸,地下部位10种成分平均含量缬草三酯>异绿原酸A>橙皮苷>绿原酸>乙酰缬草三酯>异绿原酸C>异绿原酸B>蒙花苷>新绿原酸>咖啡酸;蜘蛛香不同部位酚酸类和黄酮类含量均为地上部位>地下部位,缬草三酯类含量为地下部位>地上部位;造成蜘蛛香不同部位差异的标志物为缬草三酯、蒙花苷、橙皮苷。结论 蜘蛛香各部位样品中10个成分的含量存在较大差异;蜘蛛香的非药用部位也含有大量的酚酸类、黄酮类和缬草三酯类,应充分利用,避免中药资源的浪费。