Reversed-Phase HPLC Analysis of Steviol Glycosides Isolated from Stevia rebaudiana Bertoni

High Performance Liquid Chromatography (HPLC) experiments have been performed on nine steviol glycosides namely rebaudioside A, steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A isolated from the leaves of Stevia rebaudiana using Reversed-Phase (RP) column. Using RP-HPLC method, the individual retention times for nine naturally occurring ent-kaurane diterpene glycosides of S. rebaudiana reported in JECFA have been determined at four different temperatures: 20℃, 40℃, 60℃, and 79℃. Also, calculated the relative retention times of the eight steviol glycosides steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A against the major steviol glycoside rebaudioside A. HPLC results suggested that temperatures 40℃ and 60℃ would be ideal conditions for better separation of steviol glycosides.

Single-run reversed-phase HPLC method for determining sertraline content,enantiomeric purity,and related substances in drug substance and finished product

A direct enantio-,diastereo-,and chemo-selective high-performance liquid chromatographic method was developed for determining the content,enantiomeric purity,and related substances of the chiral antidepressant drug sertraline HCl in a single chromatographic run.The separation was achieved on a chiral stationary phase based on amylose tris(3-chloro-5-methylphenylcarbamate)under reversed-phase conditions.The method was optimized by evaluating the influence of the temperature and mobile phase composition on the retention and selectivity.The application of the single-run approach allowed to baseline resolve all investigated species in less than 15 min,without using buffers or tandem-coupled columns.The chromatographic method was validated according to the guidelines of the Official Medicines Control Laboratory and applied to control the content of sertraline HCl and related chiral substances in a generic antidepressant formulation.

HPTLC联用像素与光密度法快速定量茶饮料中的EGCG

以HPTLC分析平台,分别用像素分析法和光密度法,建立并对比两种快速检测茶饮料中EGCG含量的方法。以3种市售茶饮料为代表性样品,对比两种方法的应用性能,评估像素分析替代仪器依赖的光密度检测作为茶饮料中EGCG含量检测的新方法的先进性。结果显示,HPTLC-像素分析方法、HPTLC-光密度方法检测的定量限分别为6.8,5 mg/kg,均可以定量茶饮料中EGCG。两种方法目标物在200~1200 ng/斑点浓度范围内线性相关系数相差不大,均在0.999以上。两种方法的回收率均满足要求,HPTLC-像素分析方法测定3种样品加标回收率分别在102.4%~110.8%,102.3%~118.8%,100.3%~113.5%之间,HPTLC-光密度方法测定3种样品加标回收率分别在:103.5%~110.8%,95.3%~111.2%,99.3%~111.2%之间,表明两种方法具有良好的准确性。两种方法的精密度也相差不大,HPTLC-像素分析方法RSD<6.7%,HPTLC-光密度方法RSD<3.2%。结论:HPTLC检测技术中,简单易用的像素分析可以较好地代替昂贵的仪器化光密度扫描,提高方法的性价比和实用性。

A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

INTERSECTION POINT RULE OF THE RETENTION VALUE AND NORMAL BOILING POINT OF THE HOMOLOGUES IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

In this paper, a linear relationship between the logarithm of capacity factor k and normal boiling point to of the homologues has been derived, based on the basic retention equation of liquid chromatography according to statistical thermodyoamics proposed by professor Ln Peizhang and others, This equation has been verified by a large number of experimental data, all the strsight lines of lnk- of bumologues for different mobile phass coaiposltion cross each other at the same point, So the intereection point equation van proposed, wbich was used to prodict the retention valu, the result was satisfactory.

INTERSECTION POINT RULE OF THE RETENTION VALUE AND MOBILE PHASE COMPOSITION OF THE HOMOLOGUES IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Based on the intersection point rule of the retention value and normal boiling point of homologues in reversed-phase high-performance liquid chromatography(RPLC), the intersection point rule of the retention value of homologues and mobile phase composition has been derived, and was testified by a lot of experimental data from the literature. With this newly proposed equation, we can use the retention value of the compound in one mobile phase composition to predict its retention value in any other mobile phase composition. For fourteen groups of homologues in five mobile phase compositions on five Kinds of columns, the overall average absolute error of 721 data sets is 2.8%.

Actional Mechanism of Trifluoroacetic Acid for the Separation of Biopolymers by Reversed-phase Liquid Chromatography

The present paper covers the actional mechanism of trifluoroacetic acid for the separation of biopolymers investigated by using the parameters of stoichiometric displacement model for retention(SDM-R) in reversed-phase liquid chromatography. It was found that the trifluoroacetic acid(TFA) may participate in, or stimulate the association among displacing agent molecules in mobile phase, and decrease the affinity of both the associate molecules of the displacing agent and the TFA-protein ion-pairing. The former dominates over the separation selectivity of biopolymers as the concentration of TFA is lower than a given value, and the two contrary functions partly offset to each other and the latter dominates as its concentration is greater than the given value.

Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine

AIM:To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography(RP-HPLC),and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure(IOP)model.METHODS:The eyes of albino rabbits were administered topically and ipsilaterally with 0.2%moxonidine.A RPHPLC method was employed for the identification and quantification of moxonidine between 2 and 480 min,which presented in the aqueous humor and iris-ciliary body.Flash electroretinography(F-ERG)amplitude and superoxide dismutase(SOD)level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP.Histological and ultrastructural observation underwent to analyze the changes of retinal morphology,the inner retinal layers(IRL)thickness,and retinal ganglion cell(RGC)counting.RESULTS:Moxonidine was detectable between 2 and 480 min after administration,and the peak concentration developed both in the two tissues at 30 min,0.51μg/m Lin aqueous humor and 1.03μg/g in iris-ciliary body.In comparison to control,F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15(P<0.01)in the high IOP model;SOD levels were significantly higher at all time-points(P<0.01)with a maximum level of 20.29 U/mgprot at day 15;and RGCs were significantly higher(P<0.05).CONCLUSION:Moxonidine is a viable neuroprotective agent with application to high IOP model.All layers of retina,including RGC layer,retinal nerve fiber layer and INL,are more preserved after moxonidine administration.SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.

STUDY ON THE SEPARATION AND EXTRACTION MECHANISM OF RARE EARTH ELEMENTS BY MEANS OF REVERSED-PHASE PAPER CHROMATOGRAPHY

Reversed-phase paper chromatography technique is used for study on the extraction mechanism and sep- aration of rare earth elements.As the stationary phase,chromatographic paper strips are impregnated with a solution of monomyristyl phosphoric acid (MPA) in chloroform.Mineral acids are used as developers. The effect of concentration of acids and/or salts upon R_f has been investigated.According to the re- sults of R_f values for a given rare earth element in various acids,the order of extraction ability is HCl>HNO_3>H_2SO_4.A tetrad effect is clearly observed.for the R_f value of rare earth elements.The effects of other parameters on the R_f value,such as the quantities of extractant retained by the paper and the temperature are also examined.Based on the determination of the molar ratio of MPA to rare earth elements and the number of H^+ ions released in extraction reaction,a reasonable mechanism is proposed.The mutual separation of heavy rare earth elements will be better than that of the light rare earth group because of the larger separation coefficient of the former.A mixture of Ho-Er-Tm-Lu is successfully separated by the present method.

Determination of isotretinoin in pharmaceutical formulations by reversed-phase HPLC

The development of facile and rapid quantification of biologically active biomolecules such as isotretitoin in therapeutic drugs contained in many generic formu- lations is necessary for determining their efficiency and their quality to improve the human health care. Isotretritoin finds its applications in the maintenance of epithelial tissues. Different processes to date such as normal phase HPLC, or gas chromatrography am- ong others are able to separate and quantify isote- troin. However, the extraction is quite complex and in the case of HPLC, the analysis requires long retention times. In such context, an isocratic reversed- phase high-performance liquid chromatography (HP- LC) technique coupled with an UV-vis detector is described here for easy separation and quantification of 13-cis-retinoic (isotretinoin) from soft gelatin capsule formulations. The isotretinoin was extracted from three different commercial drug samples with tetrahydrofuran (THF) solvent by a procedure that can be completed in less than 10 minutes. Subsequent separation and quantification were accomplished in less than 5 minutes under isocratic reversed-phase conditions on a Lichrospher RP18 column and a mobile phase consisting of 0.01% TFA/acetonitrile (15/85, v/v) at a flow rate of 1.0 mL/min. Isotretoin was detected for the three samples via its UV-vis absorbance at 342 nm. The method was validated and the results showed good linearity, precision and accuracy for sensitive and selective quantitative determination of isotretinoin in the different pharmaceutical formulations. We found that the average isotretinoin content in two of the three commercial pro- ducts fell outside the 90-110% United States Pha- rmacopeia specifications. Consequently, the facile extraction and the precise method for the biomole- cule quantification open up tremendous possibilities in improving the quality control of drugs which can exist as different generic brands.

野葛根中葛根素的提取及HPTLC测定

采用有机溶剂萃取结合酸水解的方法 ,直接从野葛根中分离葛根素 ,并用 HPTL C进行纯度分析。产率为1.15 % ,含量为 97.6 %。操作简便 ,产品纯度和产率高 。

3种五味子药材HPTLC鉴别与HPLC含量测定

目的建立五味子HPTLC定性鉴别和HPLC含量测定方法。方法以五味子甲素、五味子乙素、五味子醇甲、五味子酯甲为对照品,采用HPTLC法带状点样于GF254板上,以石油醚(30～60℃)-甲酸乙酯-甲酸(15∶5∶1)为展开剂,上行展开8 cm,置于紫外灯254nm下观察,比较不同来源的五味子活性成分的差异。以五味子醇甲为对照品,采用HPLC法,ZORBAX SB-C18(4.6 mm×250 mm,5μm),流动相:甲醇-水(65∶35);流速:1.0 ml.min-1;柱温:室温;检测波长:254 nm,比较不同来源的五味子醇甲的含量。结果 HPTLC法能够准确快速鉴别不同来源的五味子;3种药材中的五味子醇甲含量有差异。结论该方法简便、准确、重现性好,可从定性、定量两方面评价五味子药材质量。

芹菜提取物中芹菜素的HPLC与HPTLC定量分析研究

通过采用高效液相色谱(HPLC)、紫外光谱和黄酮颜色特征反应对芹菜提取物主要成分芹菜素进行定性鉴定,同时建立定量测量芹菜素的高效薄层色谱(HPTLC)方法。结果表明:芹菜提取物的主要成分是黄酮醇-芹菜素,与芹菜素标准品具有相同的光谱和色谱性质;采用G60高效硅胶板,以氯仿-甲醇-水(18:2.3:0.35)为展开剂,芹菜素标样与试样有相同的Rf值为0.65;用最小二乘法作线性回归,芹菜素含量与斑点峰面积的标准曲线回归方程:y=15138x+7594.8,R2=0.993,线性范围:0.332～0.996μg/斑点。精密度实验RSD(n=5)=1.33%,平均回收率97.82%。

HPTLC法测定芹菜提取物中芹菜素的含量

本研究建立了测定芹菜素的高效薄层色谱（HPTLC）法。采用CAMAG全自动薄层色谱仪，高效硅胶G60薄层板，展开剂为氯仿-甲醇-水（18：2．3：0．35），测定波长为345nm，测定了芹菜提取物中芹菜素含量，在0．116～0．696μg／ml范围内呈良好的线性关系（R^2〉0．99）。该方法简单、快速，分离度好，灵敏度高，适用于芹菜提取物中芹菜素的测定。

HPTLC法测定庆大霉素组分及其相关物质

目的:建立测定庆大霉素组分及其相关物质的 HPTLC 法。方法:用高效薄层色谱法测定庆大霉素组分及其相关物质。CAMAG 全自动薄层色谱仪;薄层板采用 MEREK 高效硅胶 G 板,展开剂为氯仿-甲醇-25%氨水(5:7:6),测定波长为485nm。结果:庆大霉素在3.98～39.8μg·mL^(-1)。范围内各组分呈良好的线性关系(r>0.99);检测限为 ng 级。庆大霉素注射液中组分及其相关小组分之间分离度良好。结论:该方法简单、快速,灵敏度高,适用于庆大霉素组分中相关物质的分离测定。

HPTLC法测定绿茶脂溶性色素

采用丙酮∶石油醚(体积比3∶2)混合提取液直接浸提绿茶中的脂溶性色素,用石油醚∶丙酮(体积比3∶1)溶剂系统层析展开,使用CAMAG高效薄层扫描仪在450和700 nm双波长扫描脂溶性色素,结果表明,直接浸提脂溶性色素方法简便,稳定;丙酮与石油醚混合的溶剂展开系统分离清晰色素斑点13个,根据其最大吸收峰波长和Rf值确定各斑点分别为β-胡萝卜素,γ-胡萝卜素,脱镁叶绿素酸酯a,b,脱镁叶绿素a,b,玉米黄素,紫黄质,叶绿素酸酯a,叶绿素a,叶绿素酸酯b,叶绿素b.

银杏聚戊烯醇同系物的提取及其HPTLC法的初步分析

目的 :确定银杏聚戊烯醇各同系物的链长。方法 :从银杏叶的石油醚提取物中分离获得了纯度较高的聚戊烯醇同系物的混合物。进而利用反相高效薄层色谱 ,以丙酮为展开剂 ,碘蒸气显色后呈显五个斑点。结果 :经与标准品对照 ,可确定为聚戊烯醇 16～聚戊烯醇 2 0五个单体。结论 :用HPTLC能快速、简便地确定各同系物链长 ,较HPLC方法费用低廉。

农药土壤光解与HPTLC技术的应用

农药土壤光解是化学农药在土壤环境中消失的重要途径。土壤的非匀质相分布和吸附态分子的特征给农药土壤光解的研究带来很多困难,多年来对农药土壤光解并没有形成一种成熟的方法。HPTLC技术在研究农药吸附态光解上有很多优点,是一种很好的研究土壤中农药吸附态光化学行为的实验室方法。

复方补乌糖浆的HPTLC图谱研究

目的研究复方补乌糖浆(补骨脂、川芎、当归、生首乌等)高效薄层色谱,用于质量控制。方法复方补乌糖浆用碳酸钠溶液溶解,乙酸乙酯和正丁醇萃取,不同极性部位进行特征图谱研究,用Chromafinger软件进行相似度分析。结果对10批复方补乌糖浆进行了考察,乙酸乙酯部位确定了12个共有峰,各批样品特征图谱相似度均大于0.99;正丁醇部位确定了10个共有峰,各批样品特征图谱相似度均大于0.97,共有峰中包括补骨脂素、异补骨脂素、大黄素、丹参酮ⅡA、丹酚酸B和阿魏酸等6个色谱峰。结论此方法未发现复方补乌糖浆中左旋紫草素、齐墩果酸和藁本内酯。

3种新烟碱类杀虫剂在蜂蜜中HPTLC残留分析方法的研究

建立了吡虫啉、噻虫胺和噻虫嗪在蜂蜜中残留的高效薄层色谱(HPTLC)检测方法,用乙腈机械震荡提取后涡旋并离心样品,于C-18固相萃取柱净化,CAMAGLinomatⅣ点样仪点样,二氯甲烷∶乙酸乙酯=3∶1(V∶V)混合溶液单向上行展开,于254nm波长进行HPTLC检测。结果表明,当3种农药在蜂蜜中的添加浓度分别0.05、0.5和1.0 mg·kg^(-1)时,添加回收率分别为71.58%~101.25%、72.29%~109.47%和79.88%~108.92%,相对标准偏差分别为9.31%~14.47%、7.79%~12.86%和9.24%~14.23%,均满足农残分析的基本要求。