Qualitative analysis of catechins from green tea GMB-4 clone using HPLC and LC-MS/MS

Objective:To identify the bioactive compounds in catechins isolation and its components from green tea GMB-4 clone.Methods:Green tea GMB-4 clones were extracted with distilled water at 90C.Samples were eluted into the column with 10%ethanol.Subsequently,the column was eluted with95%ethanol and evaporated separately.Green tea extract was identified by thin layer chromatography.Catechins were separated by the stationary phase in column chromatography using polyamide with 10%ethanol eluent and 95%ethanol.The results of isolations were analyzed by high performance liquid chromatographic(HPLC)and LCMS/MS.Analysis of catechins by HPLC was done by external standard.Results:Fraction from 10%ethanol showed that four major peaks at retention time of1.663,2.367,2.950 and 4.890,indicated the presence of four catechins components including catechin,epicatechins,gallocatechin and epigallocatechin.Whereas,fraction from 95%ethanol showed two main peaks at retention time of 5.167 and 9.82,which indicated the presence of epigallocatechin gallate(EGCG)and epicatechin gallate(ECG).EGCG(m/z 459),epigallocatechin(m/z 307),ECG(m/z 443),and epicatechin(m/z 291)were isolated and separated successfully using HPLC and LC-MS/MS.Conclusions:The HPLC and LC-MS/MS methods were successfully tuned for the qualitative analysis of green tea extract with EGCG and ECG.Four major catechins were separated and identified by LC-MS/MS,such as EGCG,epigallocatechin,ECG and epicatechin.The result of HPLC analysis showed that EGCG and ECG were main components from catechins isolation of green tea GMB-4 clone.

Determination and pharmacokinetic study of catechin in rat plasma by HPLC

A high performance liquid chromatographic method was developed and validated for the quantitative determination of catechin in rat plasma and its pharmacokinetic study after intragastric administration of Catechu and Xiongdanjiangre Wan into SD rats. Plasma samples were prepared by protein precipitation using methanol-5% aqueous zinc sulfate （70：30, v/v） as precipitant. Chromatographic separation was achieved on Hypersil Cl8 column （250 mm~ 4.6 mm, 10 pm） with acetonitrile-water-triethylamine （6：94：0.3, v/v/v, pH 4.0＋0.1, adjusted with phos- phoric acid） as mobile phase, followed by a UV detection at 207 nm. Good linearity was obtained over the range of 0.143-7.15 mg/L of catechin, with correlation coefficient of 0.9992. The method was simple, sensitive, accurate and reproducible and＇ has been successfully applied to the pharmacokinetic study of catechin in rat plasma.

Quantitation of the Total Catechin Content in Oils Extracted from Seeds of Selected Tea (<i>Camellia sinensis</i>(L) O. Kuntze, Theaceae) Clones by RP-HPLC

Catechins (flavan-3-ols) are polyphenolic plant secondary metabolites that have been strongly associated with a wide variety of beneficial health effects in vitro, in vivo and clinically. This study reports findings on the content of catechins in tea seed oil (TSO) extracted by Soxhlet extraction from seeds of different clones of Kenyan tea. Extraction of catechins from the crude oils was achieved by sequential liquid-liquid extraction (LLE) using methanol and quantified by reverse phase High Performance Liquid Chromatography (RP-HPLC). Results obtained revealed that all the crude test oils contained catechins, with oils extracted from clones TRFK K-Purple and GW-Ejulu having the highest total catechin content of 9.8 ± 0.25 and 9.0 ± 0.83 (×10-3% flavonoids) respectively. Statistically significant differences (p < 0.05) were evident in the total catechin contents of crude oils extracted from tea seeds with those extracted from corn, sunflower and soybean seeds. Moreover, clonal variations were evident, as the total catechin contents of oils extracted from clones TRFK K-Purple and GW-Ejulu were statistically different (p < 0.05) from those extracted from clones TRFK 301/3, TRFK 301/4, TRFK 301/5, TRFK 306, TRFK 91/1 and TRFCA SFS 150. Thus, the current findings strongly suggest that oils from seeds of Kenyan tea cultivars can be a potential source of potent natural antioxidants.

Tyrosinase Biosensor Used for the Determination of Catechin Derivatives in Tea: Correlation with HPLC/DAD Method

Tea, one of the most popular consumed worldwide beverages, is known to be rich in polyphenols, more particularly in catechins. An amperometric biosensor based on tyrosinase was developed in order to monitor the phenolic content in black and green teas. The enzyme was immobilised by coreticulation with glutaraldehyde on carbon screen-printed electrodes (CSPE). Initially, the performances of the biosensor were evaluated referring to catechol as a model substrate. This analytical tool exhibits a high sensitivity (217 nA/μM), low limit of detection (LOD) = 0.03 μM, good intra-electrode and inter-electrode reproducibilities with RSD lower than 3% (n = 5 injections) and RSD = 8.14% (n = 12 sensors) respectively. The storage stability was also studied;the biosensor retained successively 85% and 70% of its initial response after 34 and 53 days. Subsequently, several catechin derivatives frequently found in teas were tested and classified relatively to their sensitivities. For tea samples, the results obtained with the biosensors were compared to high performance liquid chromatography (HPLC) analysis. A good correlation between the two methods was obtained. The calculated recovery was between 90% and 96%, proving that the proposed tyrosinase biosensor can be an alternative analytical tool for global determination of catechin derivatives in tea.

Metabolic profile analysis based on GC-TOF/MS and HPLC reveals the negative correlation between catechins and fatty acids in the cottonseed of Gossypium hirsutum

Background: The diversified and high value-added utilization of cotton by products can promote the sustainable development of modern agriculture. Di erences in potential nutrients among varieties can be explained by variations in the composition and abundance of fatty acids, polyphenols, carbohydrates, amino acids, and organic acids. Therefore, the analysis of metabolite species and relationships in cottonseed is meaningful for the development of cotton byproducts.Results: In this study, the metabolomes of three representative cotton cultivars of di erent species were compared using untargeted GC-TOF/MS analysis. A total of 263 metabolites were identified from 705 peaks, and their levels were compared across cultivars. Principal component analysis and OPLS-DA clearly distinguish these samples based on metabolites. There were significant di erences in the contents of amino acids, carbohydrates, organic acids, flavonoids, and lipids in G. hirsutum TM-1 compared with G. arboreum Shixiya1 and G. barbadense Hai7124. Notably, the bioactive nutrient compound catechin obtained from the di erential metabolites significantly accumulated in TM-1. Furthermore, a comprehensive analysis using catechin and oil-related traits was conducted in core collections of Gossypium hirsutum. The results revealed the reliability of the GC-TOF/MS analysis, as well as that catechin content has a negative association with myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, and total fatty acids.Conclusion: These findings suggest that untargeted GC-TOF/MS analysis could provide a new method for investigating the underlying plant biochemistry of nutrient variation in cottonseed, and that catechin content has a negative association with oil-related traits in cottonseed. This study may pave the way to exploit the value of cotton byproducts.

HPLC同时测定甜高粱中10种黄酮类化合物含量及抗氧化活性对比研究

试验旨在建立同时测定甜高粱不同部位中10种黄酮类化合物含量的高效液相色谱(HPLC)检测方法,并对总黄酮提取物抗氧化能力进行评价。试验以甜高粱陇甜粱2号成熟期籽粒、茎秆及全株为研究对象制备总黄酮提取物,利用HPLC同时测定不同部位总黄酮提取物中的儿茶素、氯化芹菜定、花旗松素、芦丁、槲皮素、木犀草素、柚皮素、高北美圣草素、芹菜素及麦黄酮含量。结果显示,10种黄酮类化合物分别在各自线性范围内与色谱峰峰面积线性关系良好,R^(2)为0.9984~0.9999,平均加样回收率为95.0%~105.8%,RSD为1.03%~2.86%。抗氧化性测定结果表明,甜高粱不同部位总黄酮提取物对羟基自由基、超氧阴离子自由基、1,1-二苯基-2-三硝基苯肼(DPPH)自由基和2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS^(+))自由基均具有一定的清除能力,不同浓度下清除率均表现为籽粒>全株>茎秆;籽粒总黄酮提取物浓度为1.00 g/L时,籽粒总黄酮提取物对DPPH自由基的清除率高于VC和2,6-二叔丁基-4-甲基苯酚(BHT)。相关性分析结果表明,不同部位总黄酮提取物含量与不同自由基清除率及总还原能力(FRAP)均呈正相关。研究表明,该试验方法简便,结果准确性较高,可用于甜高粱中黄酮类化合物含量的测定;甜高粱不同部位总黄酮提取物具有抗氧化活性。

HPLC及LC-MS测定榛子中氯吡苯脲和多菌灵残留

本文采用高效液相色谱法(HPLC)和液相色谱-质谱法(LC-MS)对干果榛子中氯吡苯脲和多菌灵残留量进行含量测定.HPLC和LC-MS均使用C18色谱柱,含量测定采取等度洗脱,流动相:甲醇-水(55∶45,体积比);体积流量:1.0 mL·min-1;测定波长:260 nm;柱温:30℃.LC-MS采取电喷雾离子源,梯度洗脱,体积流量:0.6 mL·min-1,柱温:30℃.结果表明,榛子中氯吡苯脲与多菌灵存在残留,氯吡苯脲质量浓度在1.00~10.00μg·mL-1范围内线性关系良好,加样回收率在95.58%~100.58%;多菌灵质量浓度在1.005~15.075μg·mL-1范围内具有良好的线性关系,加样回收率在95.61%~104.39%.实验证明,HPLC与LC-MS相结合的方法具有操作简便、灵敏度高、检出限低等优点,能有效地检测到榛子样品中膨大剂氯吡苯脲及杀菌剂多菌灵的残留,并确定其残留量,线性关系和回收率结果均令人满意.根据被检测的8批样品中氯吡苯脲和多菌灵两项农药残留量推断,作为一般干果食用榛子是安全的.

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。

HPLC测定吗替麦考酚酯口服混悬剂(骁悉)中尼泊金甲酯含量

建立了一种测定吗替麦考酚酯口服混悬剂中尼泊金甲酯含量的方法。采用高效液相色谱法,色谱柱:YMC-Pack Pro C18(150 mm×4.6 nm,5μm);流动相:甲醇-1%乙酸水(60∶40);检测波长:254 nm;流速:0.7 mL·min^(-1);柱温:30℃;进样量:10μL。采用该法测定,专属性强,精密度好(RSD=1.16%,n=12),准确度高(平均加样回收率为99.35%,回收率RSD<2%),操作简单,适用吗替麦考酚酯混悬剂生产中尼泊金甲酯含量检测。该法测得的吗替麦考酚酯混悬液中尼泊金甲酯含量为3.18 mg·mL^(-1),占混悬剂质量的0.50%。

HPLC法同时测定驻春胶囊中4个成分的含量

目的:建立以高效液相色谱法(HPLC)同时测定驻春胶囊中异补骨脂素、补骨脂素、淫羊藿苷、蛇床子素含量的方法。方法:采用HPLC法,以异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素作为指标成分,采用Agilent ZORBAX SB-C_(18)色谱柱(4.6 mm×250 mm,5μm);流动相为甲醇-水,梯度洗脱,体积流量为1.0 m·min^(-1);柱温为30℃,检测波长为254 nm,进样量为5μL。结果:异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素质量浓度分别在0.0059~0.1770 mg/mL、0.0049~0.1470 mg/mL、0.0031~0.093 mg/mL和0.0048~0.1440 mg/mL范围内与峰面积呈良好的线性关系,其相关系数分别为0.9996、0.9995、0.9997和0.9995,平均加样回收率分别为101.45%、100.95%、100.46%和101.17%;RSD分别为2.19%、1.83%、1.71%和2.01%。结论:该分析方法适用于驻春胶囊中异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素的含量测定。

HPLC法同时测定益肾泄浊合剂中5种成分的含量

目的建立HPLC法同时测定益肾泄浊合剂中没食子酸、莫诺苷、马钱苷、毛蕊异黄酮苷、大黄酸的含量。方法分析采用Agilent Eclipse XDB-C_(18)色谱柱(250 mm×4.6 mm,5μm);流动相0.1%磷酸-乙腈,梯度洗脱;体积流量1.0 mL/min;柱温30℃;检测波长254 nm。结果5种成分在各自范围内线性关系良好(r≥0.9996),平均加样回收率97.28%~101.93%,RSD 2.56%~2.84%。结论该方法准确可靠,可用于益肾泄浊合剂的质量控制。

HPLC法同时测定舒肝利胆丸中5种成分的含量

目的建立HPLC法同时测定舒肝利胆丸中甘草苷、橙皮苷、迷迭香酸、槲皮素、柴胡皂苷a的含量。方法分析采用Waters Symmetry Shield C_(18)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20℃;检测波长210、237、283、330、368 nm。结果甘草苷、橙皮苷、迷迭香酸、槲皮素、柴胡皂苷a在各自范围内线性关系良好(r>0.9995),平均加样回收率98.41%~102.42%,RSD 1.78~2.81%。结论该方法准确、稳定、快速,可用于舒肝利胆丸的质量控制。

HPLC与紫外-可见分光光度法对翻白草中黄酮类成分含量测定

目的 建立测定翻白草中黄酮类成分含量的最佳方法。方法 先建立高效液相色谱法(HPLC)测定翻白草中的4种黄酮类物质含量的方法,其中流动相为乙腈与0.1%甲酸溶液,并采用梯度洗脱,柱温设为30℃,色谱柱为InertSustain C18(150 mm×4.6 mm, 5μm),检测波长设为360 nm,并对不同批次的翻白草中的4种黄酮类物质含量进行检测;选取芦丁作为对照品,以UV-VIS法测定翻白草中总黄酮含量。结果 不同产地的翻白草含有的黄酮类成分含量差异较大,总黄酮含量的差异也较大,建立的HPLC法与紫外-可见分光光变法(UV-VIS)法具有很好地重复性,样品稳定性好,且仪器精密度高,两种检测方法相结合能很好的把控翻白草品质。结论 以UV-VIS法对翻白草中的总黄酮进行测定,并采用HPLC法测定翻白草中含有的槲皮苷、芦丁、山柰酚和槲皮素,方法较为简便,且有很好的重复性、稳定性,精密度高,能很好地控制翻白草的品质,并为临床研究及其药理活性研究提供依据。

不同产地巴戟天HPLC指纹图谱及化学模式识别研究

目的:利用HPLC指纹图谱及化学模式识别对巴戟天药材进行评价。方法:采用Shodex Asahipak NH2P-504E色谱柱(4.6 mm×250 mm,5μm),以乙腈-水为流动相梯度洗脱,使用蒸发光散射检测器(ELSD)。利用相似度评价、聚类分析和主成分分析等化学模式识别方法,对不同产地的巴戟天药材进行分析。结果:所建立的HPLC指纹图谱共识别了18个共有峰,经与对照品图谱比较,鉴定其中的4个共有峰分别为果糖、蔗糖、蔗果三糖和耐斯糖。6个产地巴戟天的相似度在0.861~0.995;经聚类分析和主成分分析,6个产地巴戟天共聚为3类。结论:本研究采用的巴戟天HPLC指纹图谱及化学模式识别技术可反映出不同产地的巴戟天样品的差异,可为巴戟天的质量控制提供参考。

HPLC法测定五种产地地龙中次黄嘌呤和肌苷的含量

目的:建立地龙中次黄嘌呤与肌苷含量的测定方法,测定五种产地地龙成分,为地龙成分的提取工艺研究、质量分析探索新型高效的研究方法。方法:建立HPLC高效液相色谱法测定分析方法。色谱柱Agilent extend C18色谱柱,4.6 mm×250 mm,粒径:5μm。流动相为甲醇-0.2%磷酸,波长选择254 nm,进样量选择20μL,柱温为30℃,使用梯度洗脱法,建立次黄嘌呤和肌苷的标准曲线,进行精密度试验,稳定性试验,重复性试验,加样回收试验等方法学考察。结果:次黄嘌呤标准曲线:y=78.819x+245.92,R2=0.9999,在10.08~161.28μg·mL^(-1)范围内呈良好线性关系;肌苷标准曲线:y=56.566x+68.333,R2=0.9995,在20.08~321.28μg·mL^(-1)范围内呈良好线性关系。实验精密度、稳定性、重复性良好,计算得到次黄嘌呤的平均回收率为95.43%,RSD值为1.52%(n=6);肌苷的平均回收率为99.86%,RSD值为2.36%(n=6),广东、河南、海南、上海、广西五种产地地龙的次黄嘌呤含量分别为:0.5335、0.3468、0.6874、0.5694、0.7945 mg·g^(-1);肌苷含量为:1.5625、0.5674、1.1267、0.8436、1.2849 mg·g^(-1)。结论:建立的HPLC高效液相色谱法,梯度洗脱效果较好,实验准确度良好,精密度良好,稳定性良好,重复性高,是一种高效的分析测定方法。

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。

HPLC法测定陕产青牛胆中药根碱、盐酸巴马汀和古伦宾的含量及质量评价

本研究通过建立高效液相色谱法(HPLC)测定陕产青牛胆中药根碱、盐酸巴马汀和古伦宾的含量,并对陕产青牛胆样品进行初步质量评价。结果表明:所收集的5批青牛胆样品(QND1~QND5)药根碱含量分别为0.33、0.12、0.14、0.53和0.08 mg/g;盐酸巴马汀含量分别为0.81、0.54、0.36、0.62和0.29 mg/g;古伦宾含量分别为10.98、7.39、5.29、4.55和0.69 mg/g。本研究建立的HPLC法可为市售青牛胆中生物碱、二萜类物质的含量检测及陕产青牛胆的质量评价提供参考依据。

巴芪柔肝方基准样品HPLC特征图谱建立及其量值传递规律研究

目的建立巴芪柔肝方基准样品HPLC特征图谱,并考察其量值传递规律。方法测定毛蕊异黄酮葡萄糖苷、橙皮苷、迷迭香酸、莪术烯醇、耐斯糖含量,计算饮片-水煎液-基准样品转移率,记录出膏率、pH值。结果15批基准样品HPLC特征图谱中有16个特征峰,相似度均大于0.90,鉴定出9个。饮片-基准样品中耐斯糖、毛蕊异黄酮葡萄糖苷平均转移率分别为(83.14±6.25)%、(77.81±8.31)%,水煎液-基准样品中迷迭香酸、莪术烯醇平均转移率分别为(81.71±6.27)%、(72.16±5.91)%,平均出膏率为(38.91±1.46)%,pH值为5.13±0.08。结论该方法稳定可行,可为巴芪柔肝方制剂工艺选择、关键化学属性评价提供参考。

清脂化瘀方HPLC-CAD特征图谱建立及6种糖类含量测定

目的建立清脂化瘀方HPLC-CAD特征图谱,并测定鼠李糖、果糖、甘露醇、D-无水葡萄糖、蔗糖、棉子糖的含量。方法分析采用Dikma Polyamino HILIC色谱柱(4.6 mm×250 mm,5μm);流动相乙腈-水,梯度洗脱;体积流量0.8 mL/min;柱温35℃。结果15批样品特征图谱中有14个特征峰相似度大于0.90。6种糖类在各自范围内线性关系良好(r>0.9950),平均加样回收率93.34%~105.64%,RSD 0.5%~2.6%。结论该方法准确可靠,灵敏度高,重复性好,可用于清脂化瘀方糖类的质量控制。