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%。     

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。     

HPLC法同时测定24种花中的5种活性成分含量

建立了高效液相色谱法(HPLC)同时测定24种花中绿原酸、金丝桃苷、槲皮素、木犀草素和异鼠李素含量的方法。采用C18柱(250 mm×4.6 mm,5μm),流动相为甲醇-0.2%冰乙酸溶液梯度洗脱,检测波长350 nm,柱温35℃,流速0.8 mL/min。绿原酸、金丝桃苷、槲皮素、木犀草素和异鼠李素分别在0.0208~104.00μg/mL(r=0.99993),0.017~85.00μg/mL(r=0.99998),0.0172~86.00μg/mL(r=0.99997),0.0304~152.00μg/mL(r=0.99997),0.0168~84.00μg/mL(r=0.99986)范围内与峰面积呈良好的线性关系,平均加标回收率(n=9)92.26%~99.09%,仪器精密度(n=6)RSD均小于3.1%,方法重复性(n=6)的RSD均小于3.6%。方法可同时测定这24种花中绿原酸和黄酮类物质的含量,可作为花中活性成分定量分析的方法。     

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。     

高效液相色谱法测定润肤霜和洗发露中的银杏黄酮类物质

建立了测定润肤霜和洗发露中3种银杏黄酮类物质(槲皮素、山萘素和异鼠李素)的高效液相色谱分析方法。样品经甲醇提取、离心后,提取液以Fortis Phenyl色谱柱(4.6×250 mm,5μm)分离,体积分数0.5%H3PO4-甲醇流动相体系等度洗脱,在波长367 nm下用二极管阵列检测器检测,以保留时间结合紫外光谱定性,外标法定量。结果表明:3种目标分析物分离度好;在0.25~50.0 mg/L范围内线性关系良好,相关系数为0.9992~0.999 9;槲皮素、山萘素和异鼠李素的方法检出限(S/N=3)分别为0.8,1.0和1.0 mg/kg;添加水平为5~500 mg/kg时,回收率为83.5%~102.8%,相对标准偏差(RSD,n=6)为1.6%~6.5%。方法适用于护肤类和洗护发类化妆品中槲皮素、山萘素和异鼠李素的测定。     

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。     

高效液相色谱法同时检测4种金线莲黄酮苷元及其在提取工艺评价中的应用

基于高效液相色谱(HPLC)和提取离子流色谱-质谱(EIC-MS),建立了金线莲提取成分定性定量分析及提取效率评价新方法。方法采用20RBAX Ecipse XDB-C18色谱柱,以乙腈-0.03%乙酸水溶液为流动相,采用梯度洗脱,同时分离测定金线莲提取液中芦丁、山奈酚、异鼠李素和槲皮素4种黄酮苷元的含量,经EIC-MS验证,结果可靠。以黄酮提取率为指标,评价酶解-醇提、回流-醇提、搅拌-醇提、超声-醇提、超声-微波-醇提和煎煮6种提取工艺,结果显示,最佳提取工艺为回流-醇提法,提取时间短,醇用量少,总提取率最高,可达14.96mg/g。     

高效液相色谱法测定野葱中黄酮类化合物

建立了测定野葱中芦丁、黄酮醇类化合物的高效液相色谱方法。80%乙醇超声提取,高效液相色谱分析野葱中芦丁和黄酮醇类化合物的含量。芦丁检测条件:V(甲醇)∶V(0.2%磷酸水)=45∶55,检测波长:360nm;槲皮素、山奈酚、异鼠李素检测条件:V(甲醇)∶V(0.2%磷酸水)=40∶60,检测波长:360 nm。结果表明,野葱中芦丁含量0.22%,槲皮素、山奈酚、异鼠李素含量依次为0.42%、0、0.23%,总黄酮醇类化合物含量为1.63%。     

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%。     

High-performance liquid chromatographic fingerprint analysis for different origins of sea buckthorn berries

Using high-performance liquid chromatography (HPLC), a chemical fingerprint method was developed for investigating and demonstrating the variance of flavonoids among different origins of sea buckthorn berries. Thirty-four samples were analyzed including 15 RS (Hippophae rhamnoides ssp. sinensis) samples, 7 RY (H. rhamnoindes ssp. yunnanensis) samples, 5 RW (H. rhamnoides ssp. wolongensis) samples, 4 NS (H. neurocarpa ssp. stellatopilosa) samples and 3 TI (H. tibetana) samples. In the HPLC chromatograms, 12 compounds were identified as flavonoids, including quercetin 3-O-sophoroside-7-rhamnoside, kaempferol 3-O-sophoroside-7-O-rhamnoside, isorhamnetin 3-O-sophoroside-7-O-rhamnoside, isorhamnetin 3-O-glucoside-7-O-rhamnoside, quercetin 3-O-rutinoside, quercetin 3-O-glucoside, isorhamnetin 3-O-rutinoside, isorhamnetin 3-O-glucoside, quercetin, kaempferol 7-O-rhamnoside, kaempferol and isorhamnetin. Both correlation coefficient of similarity in chromatograms and relative peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. Our results revealed that the chromatographic fingerprint combining similarity evaluation could efficiently identify and distinguish sea buckthorn berries from different species. However, no obvious difference between RS and RY suggested that the two subspecies might have very close relationship in terms of chemotaxonomy. The established method was considered to be suitable for fingerprint analysis to check the genuine origin and control the quality of sea buckthorn berries and extracts.     

油菜蜂花粉黄酮含量的HPLC测定

以95％乙醇为溶剂,采用索氏提取器提取青海产油菜蜂花粉中的黄酮类化合物,将黄酮提取物中的黄酮甙水解为黄酮甙元后,利用HPLC法测定其中槲皮素、山萘酚、异鼠李素含量。结果表明,青海油菜蜂花粉中槲皮素、山萘酚、异鼠李素的平均含量分别为0．928％、0．295％、0．0834％,换算成总黄酮含量为3．28％。     

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.     

高效液相色谱-串联质谱法同时测定银杏保健茶中的16种黄酮类功效成分

建立了银杏保健茶中16种黄酮类物质的液相色谱-串联质谱(LC-MS/MS)测定方法。16种黄酮成分分别为儿茶素、牡荆素、葛根素、大豆苷元、水飞蓟宾、槲皮素、木犀草素、芹菜素、柚皮素、橙皮素二氢查尔酮、山柰酚、橙皮素、异鼠李素、黄芩素、川陈皮素、桔皮素。实验优化了液相色谱条件和质谱参数。采用C₁₈柱分离,流动相为乙腈-水(含0.1%甲酸)梯度洗脱,流速0.25 mL/min,以电喷雾离子源正离子多反应监测(MRM)模式进行MS/MS检测。16种黄酮类物质在各自的线性范围内具有良好的线性关系,相关系数大于0.996,低、中、高3个添加水平的平均回收率在70.9%~100.0%之间,相对标准偏差小于10%。通过检测发现实际样品中9种黄酮物质含量较高,分别是:山柰酚、槲皮素、橙皮素、牡荆素、木犀草素、儿茶素、芹菜素、柚皮素、异鼠李素,占总量的99.6%,此9种物质可作为银杏保健茶的质量控制指标。本法简便、快速、准确可靠,可用于控制银杏保健茶的质量。     

Analysis of flavonoids in honey by HPLC coupled with coulometric electrode array detection and electrospray ionization mass spectrometry

The analysis of flavonoids in unifloral honeys by high-performance liquid chromatography (HPLC) coupled with coulometric electrode array detection (CEAD) is described. The compounds were extracted by a nonionic polymeric resin (Amberlite XAD-2) and then separated on a reversed phase column using gradient elution. Quercetin, naringenin, hesperetin, luteolin, kaempferol, isorhamnetin, and galangin were detected in a coulometric electrode array detection system between +300 and +800 mV against palladium reference electrodes, and their presence was additionally confirmed by HPLC coupled with electrospray ionization mass spectrometry. The method was applied to analysis of 19 honeys of different varieties and origin. The limits of detection and quantitation ranged between 1.6 and 8.3 μg/kg and 3.9 and 27.4 μg/kg, respectively. The recoveries were above 96% in fluid and above 89% in creamy honeys. Some of these honeys (melon, pumpkin, cherry blossom, dandelion, maple, and pine tree honey) were investigated for their flavonoid content and profile for the first time. Differences between honeys were observed both in flavonoid concentrations and in the flavonoid profiles. The flavonoid concentrations ranged from 0.015 to 3.4 mg/kg honey. Galangin, kaempferol, quercetin, isorhamnetin, and luteolin were detected in all investigated honeys, whereas hesperetin occurred only in lemon and orange honeys and naringenin in lemon, orange, rhododendron, rosemary, and cherry blossom honeys.     

Direct identification of phenolic constituents in Boldo Folium (Peumus boldus Mol.) infusions by high-performance liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry

A very simple and direct method was developed for the qualitative analysis of polyphenols in boldo (Peumus boldus Mol., Monimiaceae) leaves infusions by high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (HPLC-MSⁿ). The phenolic constituents identified in infusions of the crude drug Boldo Folium were mainly proanthocyanidins and flavonol glycosides. In the infusions, 41 compounds were detected in male and 43 compounds in female leaf samples, respectively. Nine quercetin glycosides, eight kaempferol derivatives, nine isorhamnetin glycosides, three phenolic acids, one caffeoylquinic acid glycoside and twenty one proanthocyanidins were identified by HPLC-DAD and ESI-MS for the first time in the crude drug. Isorhamnetin glucosyl-di-rhamnoside was the most abundant flavonol glycoside in the male boldo sample, whereas isorhamnetin di-glucosyl-di-rhamnoside was the main phenolic compound in female boldo leaves infusion. The results suggest that the medicinal properties reported for this popular infusion should be attributed not only to the presence of catechin and boldine but also to several phenolic compounds with known antioxidant activity. The HPLC fingerprint obtained can be useful in the authentication of the crude drug Boldo Folium as well as for qualitative analysis and differentiation of plant populations in the tree distribution range.     

Rapid screening and evaluation of XOD inhibitors and O2•− scavenger from total flavonoids of Ginkgo biloba leaves by LC–MS and multimode microplate reader

Superoxide anion radical scavenger and xanthine oxidase inhibitor play an important role in the treatment of several relevant human diseases. In the present study, ultrafiltration liquid chromatography–mass spectrometry coupled to microplate reader was applied to screen and identify superoxide anion radical scavengers and xanthine oxidase inhibitors from total flavonoids of Ginkgo biloba leaves. As a result, four compounds (quercetin, apigenin, kaempferol and isorhamnetin) were screened as xanthine oxidase inhibitors by ultrafiltration LC–MS, and the 50% scavenging concentration values of the screened flavonoids were lower than those for allopurinol. Lineweaver–Burk plot results indicated that kaempferol was a competitive xanthine oxidase inhibitor; the other flavonoids were all anticompetitive inhibitors. Four flavonoids—rutin, quercetin, kaempferol and isorhamnetin—were screened as superoxide anion radical scavengers by LC–MS. The results demonstrate that the method for screening and evaluation of superoxide anion radical scavenger and xanthine oxidase inhibitor from a complex mixture system is feasible and efficient.     

Quantitative determination of isorhamnetin, quercetin and kaempferol in rat plasma by liquid chromatography with electrospray ionization tandem mass spectrometry and its application to the pharmacokinetic study of isorhamnetin

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of quercetin, kaempferol and isorhamnetin in rat plasma. After being treated with beta-glucuronidase and sulfatase, the analytes were extracted by liquid/liquid extraction with the internal standard (IS; baicalein). The chromatographic separation was performed on a Diamonsil C(18) column with a mobile phase consisting of 2% formic acid/methanol (10:90, v/v) at a flow rate of 1.00 mL/min, with a split of 200 microL to the mass spectrometer. Validation results indicated that the lower limit of quantification (LLOQ) was 1 ng . mL(-1). The assay exhibited a linear range of 1-200 ng . mL(-1) and gave a correlation coefficient of 0.9980 or better for each analyte. Quality control samples (1, 5, 20 and 100 ng . mL(-1)) in six replicates from each of three different runs demonstrated an intra-assay precision (RSD) of 1.1-8.9%, an inter-assay precision of 1.6-10.8%, and an overall accuracy (bias) of <13.4%. The extraction recovery of each analyte and internal standard was 70-80%. In the present study, we have investigated the pharmacokinetic profiles of isorhamnetin after oral application in rats equipped with a jugular catheter. After oral dosing of isorhamnetin, the mean values (n = 10) of C(max) were 57.8, 64.8 and 75.2 ng . mL(-1) which were achieved at a T(max) of 8.0, 6.4 and 7.2 h for oral doses of 0.25, 0.5 and 1.0 mg . kg(-1) body weight, respectively. The corresponding mean values for isorhamnetin area under the curver (AUC) from 0 to 60 h were 838.2, 1262.8, 1623.4 ng . h . mL(-1). Our results further demonstrated that the samples analyzed showed isorhamnetin could not be transformed into quercetin or kaempferol in rats, indicating that the demethylation of the 3'-oxymethyl group of isorhamnetin does not occur in Wistar rats.     

HPLC quantification of dye flavonoids in Reseda luteola L. from Portugal

A HPLC method was developed for the simultaneous identification of Reseda luteola L. (weld) flavonoids and quantification of the main compounds responsible for the yellow color. This method was applied to a large number of wild Portuguese weld to evaluate its potential application as dyestuff for textile factories, as a substitute for the synthetic dyes currently used. Portuguese weld dyestuff content ranged between 1.04 and 5.87%, corresponding to a wide variation of the flavonoids amount (1.39–9.04%). Luteolin 4′‐O‐glucoside was found for the first time in R. luteola, but kaempferol, isorhamnetin, and their glycosides were not detected in the Portuguese specimens.     

HPLC Separation of O-Acylated Flavonoids and Biflavones from Some Species of Gymnospermae

Flavonoids present in the extracts from leaves of Pseudotsuga menziesii (Pinaceae), Ginkgo biloba (Ginkgoaceae) and Podocarpus dacrydioides (Podocarpaceae) were separated by use of the reversed phase HPLC method. The analysed compounds belong to different groups of flavonoids – biflavones (amentoflavone, bilobetin, 5–methoxybilobetin, podocarpusflavone A, sequoiaflavone, podocarpusflavone B, ginkgetin, isoginkgetin, sciadopitysin, kayaflavone, hinokiflavone, 2,3–dihydrosciadopitysin, 2,3–dihydroisoginkgetin), O–acylated flavonol glycosides (daglesiosides I, II, III, IV, trans–tiliroside, trans–ditiliroside), flavonol O–glycosides (astragalin, isoquercetin) and flavonol aglycones (kaempferol, quercetin, isorhamnetin). The conditions for flavonoid separation were optimized using various RP–18 columns. The chromatographic resolution was performed with isocratic or gradient elution – optimized by Drylab program or by traditional trial-and-error method, depending on the composition of flavonoid complex.