高效液相色谱法同时测定枸杞中槲皮素、山柰酚和异鼠李素

建立了高效液相色谱同时检测枸杞中槲皮素、山柰酚和异鼠李素的分析方法。样品经过甲醇超声提取后,用甲醇-25%HCl水解1 h,采用Inertsustain C18色谱柱进行分离,以甲醇-0.4%H3PO4溶液(48∶52,V/V)为流动相,进行等度洗脱,流速为1.0 m L/min,二极管阵列检测器检测,检测波长为360 nm,柱温为40℃。槲皮素,山柰酚,异鼠李素在40 min内实现分离,并分别在0.053~21.2μg/m L,0.053~4.24μg/m L和0.046~3.72μg/m L范围内具有良好的线性关系,相关系数为0.9972~0.9992,测得槲皮素、山柰酚、异鼠李素的加标回收率为99.2%~103.1%,95.6%~101.8%,93.2%~109.1%;相对标准偏差分别为0.95%~2.8%,0.55%~2.3%,0.81%~2.4%。对槲皮素、山柰酚和异鼠李素的检出限分别为0.04,0.05,0.03 mg/kg。方法可用来测定枸杞中3种黄酮苷元的含量。     

反相高效液相色谱法测定不同产地布渣叶的3种水解黄酮苷元

提出了反相高效液相色谱法同时测定布渣叶的3种水解黄酮苷元槲皮素、山奈酚和异鼠李素的含量。采用Kromasil C18色谱柱(250 mm×4.6 mm,5μm),以甲醇-0.4%(体积分数)磷酸溶液为流动相梯度洗脱,在368 nm波长处,对布渣叶的水解液进行了色谱分离测定。结果表明:槲皮素的质量浓度在1.97~19.7 mg.L-1,山奈酚在2.02~20.2 mg.L-1,异鼠李素在2.11~21.1 mg.L-1时分别与其峰面积呈线性关系。槲皮素、山奈酚和异鼠李素的加标回收率分别为93.0%~99.6%,92.6%~99.8%,92.5%~99.5%,相对标准偏差(n=6)分别为2.48%,2.80%,3.17%。     

HPLC法测定新疆红景天根中总黄酮的含量

建立测定红景天根中总黄酮含量的HPLC法。采用Thermo-C18(4.6 mm×200 mm,5μm)为色谱柱,V(甲醇)∶V(0.4%H3PO4溶液)=50∶50为流动相,流速1.0 mL/min,检测波长360 nm,柱温25℃。槲皮素、山奈酚、异鼠李素的线性范围分别为O.0128~0.0384、0.24~0.48及0.12~0.44μg;平均回收率分别为106.30%(RSD=1.97%)、102.30%(RSD=1.69%)和100.90%(RSD=1.62%)。所建立的HPLC法可用于测定红景天属植物中总黄酮的含量。     

高效液相色谱法同时测定不同产地及不同药用部位景天三七中4种黄酮类成分的含量北大核心CSCD

建立了同时测定不同产地及不同药用部位景天三七中槲皮素、木犀草素、山奈酚和异鼠李素含量的高效液相色谱(HPLC)法。采用TOP ODS-AQ色谱柱(250×4.6mm,5μm),以甲醇-0.1%磷酸溶液为流动相梯度洗脱,检测波长为365nm。结果表明,槲皮素、木犀草素、山奈酚和异鼠李素的浓度分别在1.90~189.90μg·mL-1(r=0.99996),1.12~112.00μg·mL-1(r=0.99998),3.71~370.56μg·mL-1(r=0.99995)和0.98~97.60μg·mL-1(r=0.99996)范围内与其色谱峰面积呈良好线性关系;平均加标回收率分别为99.79%、100.06%、100.19%和100.00%,且不同产地及不同药用部位的4个黄酮类成分在数量上或质量上有明显差异。该方法快速、准确,重现性好,可用于同时测定景天三七中槲皮素、木犀草素、山奈酚和异鼠李素含量。     

反相高效液相色谱法测定花椒的3种黄酮苷元的含量

提出了反相高效液相色谱法测定花椒的3种水解黄酮苷元槲皮素、山柰酚、异鼠李素的含量。采用Zorlbax Eclipse C_(18)色谱柱(4.6mm×150mm,5μm),以甲醇-0.4%(体积分数)磷酸溶液(1+1)混合溶液为流动相,在360nm波长处进行测定。槲皮素的质量浓度在2.19～109.6mg·L~(-1),山柰酚的质量浓度在2.28～114.0mg·L~(-1),异鼠李素的质量浓度在2.80～140.0mg·L~(-1)时分别与其峰面积呈线性关系。槲皮素、山柰酚和异鼠李素的加标回收率分别为95.6%,104.4%,103.8%,相对标准偏差(n=5)分别为2.98%,3.97%,4.30%。     

胶束电动毛细管色谱法用于中成药原料银杏浸膏中黄酮的质量控制

研究建立了胶束电动毛细管色谱法测定中药银杏浸膏(GBE)中黄酮的方法。以SDS作表面活性剂，在 25kV电压下，考察了不同缓冲体系的pH值及浓度对黄酮的3个水解产物槲皮素、山奈酚、异鼠李素分离度的影响。结果表明，选择25mmol/L硼砂-25mmol/L磷酸二氢钾—1％(V／V)甲醇电解液，15min之内槲皮素、山奈酚、异鼠李素可得到很好分离。把MECC定量分析的结果与反相高效液相色谱进行了对比，表明所建立的MECC法用于中药中的黄酮测定是可靠的。     

RP-HPLC/二极管阵列检测器同时测定飞机草中3种黄酮

应用高效液相色谱法/二极管阵列检测器同时测定了飞机草中木犀草素、槲皮素和山柰酚的含量。色谱柱HiQ sil C18W柱(4.6 mm×25 cm,5μm),流动相V(甲醇)∶V(水)∶V(磷酸)=50∶49.8∶0.2,检测波长槲皮素254 nm,木犀草素和山柰酚360 nm,温度30℃,流速1 mL/min,进样量10μL。确定了以超声波提取法制备飞机草分析样品的方法:溶剂为体积分数85%的乙醇,液固比为10∶1(mL/g),提取时间为1 h。结果表明,3种黄酮在0.01×10-3~0.10×10-3g/mL范围内呈现良好线性关系(R2>0.999 0),平均加样回收率分别为99.601 7%、99.032 6%和99.450 8%,RSD<2%。该方法操作简便、准确度高,可快速测定飞机草中木犀草素、槲皮素和山柰酚3种物质的含量。     

高效液相色谱法测定北柴胡地下部分黄酮类化合物的含量

建立了高效液相色谱法测定北柴胡地下部分黄酮类化合物含量的方法。采用SepaxGP-C18色谱柱(250×4.6 mm,5μm),流动相为乙腈-0.4%磷酸(体积比35∶65),检测波长360 nm,柱温30℃,流速1.0 mL/min。结果表明,北柴胡地下部分含有槲皮素。芦丁、木犀草素、槲皮素、山奈酚、芹菜素分别在0.0050～0.0248、0.0050～0.0248、0.0051～0.0256、0.0046～0.0232、0.0054～0.0272 mg/mL范围内线性关系良好,相关系数分别为0.9957、0.9995、0.9998、0.9998、0.9998,槲皮素的平均回收率为98.34%,相对标准偏差(RSD)为0.76%。该法简便,快速,准确,重复性好,可作为北柴胡药材质量控制的方法。     

超临界流体色谱法测定银杏叶提取物中的黄酮类化合物

采用超临界流体色谱法分离测定了银杏叶提取物水解后的三个甙元－－槲皮素、山奈酚、异鼠李素的含量。以苯基柱为固定相,二氧化碳－乙醇－磷酸９０：９．９８：０．０２Ｖ／Ｖ）为流动相,三个黄酮甙元获得良好的分离,利用各甙元的转换因子计算了黄酮化合物的总黄酮的含量,这种方法定量结果准确,重现性好。     

圆萼刺参中的芦丁,熊果酸和齐墩果酸的定性定量分析

采用薄层色谱法(TLC)和反相高效液相色谱法(RP-HPLC)对藏药材圆萼刺参中的芦丁,熊果酸和齐墩果酸进行定性和定量的分析。定性分析:TLC法检定芦丁和熊果酸和齐墩果酸成分,薄层色谱条件是以V(乙酸乙酯)∶V(丁酮)∶V(甲酸)∶V(水)=10∶6∶1∶2为展开剂,喷以10 g/L NaNO2的1%甲醇溶液在105℃检定芦丁,以V(CHCl3)∶V(乙酸乙酯)=1∶1为展开剂,喷以V(H2SO4)∶V(甲醇)=1∶2溶液在105℃检定熊果酸和齐墩果酸。定量分析:RP-HPLC法测定圆萼刺参中芦丁,流动相:V(甲醇)∶V(0.4%H3PO4溶液)=38∶62;检测波长340 nm;RP-HPLC法测定熊果酸和齐墩果酸,流动相:V(甲醇)∶V(0.2%H3PO4)溶液=85∶15;检测波长215 nm;圆萼刺参中的齐墩果酸,在本种植物中首次发现。     

高效液相色谱法测定不同产地枇杷叶中的3种黄酮类成分

建立了高效液相色谱同时测定枇杷叶中3种黄酮类成分的分析方法。该方法分析了不同产地枇杷叶中芦丁、槲皮素和山柰酚的含量差异。枇杷叶粉末用甲醇超声提取后,加盐酸回流,制备样品测试液。采用Diamonsil C18色谱柱（250 mm×4.6 mm,5 μm）,以0.4%（v/v）磷酸水溶液-乙腈为流动相,梯度洗脱。分别对7个不同产地的枇杷叶样品中的芦丁、槲皮素和山柰酚进行测定。结果表明,芦丁、槲皮素、山柰酚在各自的质量浓度范围内线性关系良好（r>0.99）,加标回收率分别为96.33%、95.81%和95.80%,RSD分别为6.48%、0.90%和3.02%。该方法操作简单、分离度好、重复性高。不同产地枇杷叶中3种黄酮类成分的含量存在差异,其中芦丁的差异最大,而山柰酚的含量最稳定且在不同产地样品中均可检出,或可用作枇杷叶药材质量控制的标志成分。     

High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Four Flavonols in Food Supplements and Pharmaceutical Formulations

A method using high-performance liquid chromatography with diode array detection (HPLC-DAD) as a powerful separation technique has been developed for the simultaneous determination of the four flavonols rutin, quercetin, kaempferol and isorhamnetin in food supplements and pharmaceutical formulations. The chromatographic separation was achieved in 36?min using a Symmetry C₁₈ column (250?×?3?mm; 5?µm) as the stationary phase and a mixture of methanol, acetonitrile, and pH 2.5 aqueous acetic acid as the mobile phase in gradient elution mode. The analytical wavelengths were 256?nm for rutin, quercetin and isorhamnetin, and 368?nm for kaempferol. An ultrasound-assisted extraction protocol was performed using methanol as solvent. The detection and quantification limits were lower than 0.03?µg mL^?1 and 0.08?µg mL^?1, respectively. The inter-day and intra-day precisions were less than 4.8 and 5.1%, respectively, and the average recoveries were in the range from 96 to 107%. The method was applied for the determination of the studied flavonols in food supplements and pharmaceutical preparations. The satisfactory recovery values demonstrate the potential of the developed method for the determination of the analytes in these samples. In addition, the method is suitable for routine quality control due its ease of operation.     

Simultaneous determination of quercetin, kaempferol, and isorhamnetin in phytopharmaceuticals of Hippophae rhamnoides L. by high-performance liquid chromatography with chemiluminescence detection

A novel method based on reversed-phase high-performance liquid chromatography with chemiluminescence detection has been developed for the simultaneous determination of three flavonols including quercetin, kaempferol, and isorhamnetin. The procedure was based on the chemiluminescent enhancement by flavonols of the cerium(IV)-rhodamine 6G system in sulfuric acid medium. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. Good separation was achieved with isocratic elution using a mixture of methanol and aqueous 1.0% acetic acid (37:63, v/v) within 25 min. Under optimized conditions, the linear working range covers 3 orders of magnitude with relative standard deviations below 4.5% for 11 replicate injected flavonol samples, and detection limits (S/N= 3) were 1.6 x 10(-8), 3.5 x 10(-9), and 6.5 x 10(-9) g mL(-1) for quercetin, kaempferol, and isorhamnetin, respectively. The chemiluminescence reaction was compatible with the mobile phase of high-performance liquid chromatography. The proposed method has been successfully applied to the determination of three active flavonols in phytopharmaceuticals of Hippophae rhamnoides L. After a simple extraction procedure, the repeatability and recovery were satisfactory.     

Molecular imprinted polymer for solid‐phase extraction of flavonol aglycones from Moringa oleifera extracts

Molecular imprinted polymer produced using quercetin as the imprinting compound was applied for the extraction of flavonol aglycones (quercetin and kaempferol) from Moringa oleifera methanolic extracts obtained using heated reflux extraction method. Identification and quantification of these flavonols in the Moringa extracts was achieved using high performance liquid chromatography with ultra violet detection. Breakthrough volume and retention capacity of molecular imprinted polymer SPE was investigated using a mixture of myricetin, quercetin and kaempferol. The calculated theoretical number of plates was found to be 14, 50 and 8 for myricetin, quercetin and kaempferol, respectively. Calculated adsorption capacities were 2.0, 3.4 and 3.7 μmol/g for myricetin, quercetin and kaempferol, respectively. No myricetin was observed in Moringa methanol extracts. Recoveries of quercetin and kaempferol from Moringa methanol extracts of leaves and flowers ranged from 77 to 85% and 75 to 86%, respectively, demonstrating the feasibility of using the developed molecularly imprinted SPE method for quantitative clean‐up of both of these flavonoids. Using heated reflux extraction combined with molecularly imprinted SPE, quercetin concentrations of 975 ± 58 and 845 ± 32 mg/kg were determined in Moringa leaves and flowers, respectively. However, the concentrations of kaempferol found in leaves and flowers were 2100 ± 176 and 2802 ± 157 mg/kg, respectively.     

Determination of seven polyphenols in water by high performance liquid chromatography combined with preconcentration

A method for determination of seven polyphenols (chlorogenic acid, esculetin, caffeic acid, scopoletin, rutin, quercetin hydrate, kaempferol) by reversed-phase high performance liquid chromatography (RP-HPLC) combined with preconcentration was developed. The preconcentration was accomplished by adsorption-desorption method with a styrene-divinylbenzene resin (XAD-4), and the analytes were desorbed by methanol. The parameters of adsorption and desorption, such as the amounts of resin, adsorption time, pH of the adsorption solution, and the volume of methanol for desorption were optimized. RP-HPLC with photodiode array detector (PAD) was employed for the qualitative and quantitative analysis. Methanol and acetic-water (1:99, v/v) solution were used as the mobile phase, and a gradient program was established for separation. Calibration curves of the seven analytes were obtained in the range of 0.8-3 mg L⁻¹, with correlation coefficients (R) higher than 0.9990. With standard samples, the recoveries for the preconcentration step under optimal conditions were 93-99%, and the relative standard deviations were 0.2-2.0% (n = 5). Polyphenols in simulated tobacco-polluted water were analyzed with the optimized conditions. Chlorogenic acid and rutin were found and determined, whose concentrations were 32.8 and 19.2 μg L⁻¹, respectively. The spiked recoveries of the polyphenols were 83-95% except quercetin hydrate (63%), the relative standard deviations were less than 3.5% (n = 5).     

Study of the reaction products of flavonols with 2,2-diphenyl-1-picrylhydrazyl using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry

The products obtained after the reaction between flavonols and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) in both methanol and acetonitrile were characterized using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and NMR spectroscopy. The flavonols studied were quercetin, kaempferol and myricetin. In methanol, two reaction products of oxidized quercetin were identified using LC/ESI-MS/MS and NMR. Quercetin was oxidized through a transfer of two H-atoms to DPPH(*) and subsequently incorporated either two CH(3)OH molecules or one CH(3)OH- and one H(2)O molecule giving the products 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dimethoxy-2,3-dihydrochromen-4-one and 2-(3,4-dihydroxyphenyl)-3,3,5,7-tetrahydroxy-2-methoxy-2,3-dihydrochromen-4-one, respectively. LC/ESI-MS/MS analysis revealed that in methanol, kaempferol and myricetin also gave rise to methoxylated oxidation products similar to that identified for quercetin. Kaempferol, in addition, also exhibited products where a kaempferol radical, obtained by a transfer of one H-atom to DPPH(*), reacted with CH(3)OH through the addition of CH(3)O(*), yielding two isomeric products. When the reaction took place in acetonitrile, LC/ESI-MS/MS analysis showed that both quercetin and myricetin formed stable isomeric quinone products obtained by a transfer of two H-atoms to DPPH(*). In contrast, kaempferol formed two isomeric products where a kaempferol radical reacted with H(2)O through the addition of OH(*), i.e. similar to the reaction of kaempferol radicals with CH(3)OH.     

Investigation of Chemiluminescence Behavior of Flavonoids with Cerium (IV)‐Rhodamine B System

Abstract

The chemiluminescence (CL) behavior of five major flavonoid types in cerium (IV)‐rhodamine B system was investigated by flow‐injection. Strong CL was observed when cerium (IV) reacted with rhodamine B in sulfuric acid medium in the presence of flavonoids. This reaction system has been established as a simple, rapid, and highly sensitive flow injection CL analysis for quercetin and kaempferol, and their detection limit (3σ) was 2.7 and 0.22 nmol/L, respectively. The relative standard deviation (n=8) was 1.2% for 1.0 µmol/L quercetin and 1.9% for 0.5 µmol/L kaempferol. This method was successfully applied to the determination of quercetin in the hydrolysate of rutin and compared well with the high performance liquid chromatography (HPLC) method. From a comparison of several related flavonoids, it was concluded that only flavonoids that contain a free 3‐hydroxyl and 2, 3‐double bond in conjugation with 4‐oxo function could produce a relatively strong CL emission.     

液相色谱法同时测定维药蜀葵花中芦丁、槲皮素和山柰酚

维药是祖国医药学不可分割的组成部分。维药现代化,即利用现代技术研究维药的有效成分,是维药科学化、标准化、规范化、商品化和产业化的必经之路。本文建立了维药蜀葵花中有效成分芦丁、槲皮素和山柰酚的选择性提取方法,优化了高效液相色谱法(HPLC)同时测定这3种有效成分的分析条件。采用HC-C₁₈色谱柱(250 mm×4.6 mm, 5 μm)和甲醇-0.4%磷酸(50:50, v/v)流动相,在柱温30 ℃和流速1.00 mL/min的条件下实现了3种物质之间以及和干扰物之间的基线分离。维药蜀葵花中芦丁、槲皮素及山柰酚的线性范围分别为12.5~150 μg/mL (r=0.9998), 12.5~125 μg/mL (r=0.9999)及12.5~125 μg/mL (r=0.9988),加标回收率(n=5)分别为100.3%(RSD=1.1%)、97.60%(RSD=0.47%)、97.75%(RSD=0.71%)。该方法实现了同时测定维药蜀葵花中芦丁、槲皮素及山柰酚,为其他黄酮类物质的开发应用提供了科学依据,同时也可为其他维药分析提供借鉴。     

Development and Validation of an SPE-LC Method for the Simultaneous Determination of trans-Resveratrol and Selected Flavonoids in Wine

A simple LC method has been developed for the simultaneous determination of the flavonols rutin, myricetin, quercetin, kaempferol and the stilbene, trans-resveratrol, in wines. Sample clean-up was performed with polymeric Nexus ABS ELUT cartridges. The polyphenols were separated in less than 25 min using gradient elution and UV detection at 320 nm. Average recoveries of the analytes from spiked wine ranged from 82.2 to 117.6% and the detection limits, based on spiked extracted synthetic wine, ranged from 0.02 to 0.2 mgL^?1. The method was applied to the analysis of Greek wines.