Counter-current chromatography based analysis of synergy in an anti-tuberculosis ethnobotanical

The crude extract of an Alaskan ethnobotanical plant, Oplopanax horridus, was subjected to counter-current chromatography (CCC), and the selected active regions were evaluated for their synergistic effects with an in vitro model of anti-tubercular efficacy. CCC as a support-free high-resolution separation method was employed to preclude potential irreversible absorption to a solid stationary phase. The microplate Alamar blue assay and the isobole method were used to measure the biological activity and eliminate dose-response dependent errors, respectively. Using the combination of CCC, bioassay and isobole method, significant synergistic effects were observed. Among the entire polarity range, fractions with distribution constant between 0.44 and 0.81 showed the most synergistic enhancement with an increase in potency by 108% for the recombined fractions.     

Overpressured layer chromatography in comparison with thin-layer and high-performance liquid chromatography for the determination of coumarins with reference to the composition of the mobile phase

The retention behaviour of fifteen closely related coumarins in normal-phase overpressured layer chromatography (OPLC) was studied with the aim of comparing the retentions with those in normal-phase thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) when optimization of the mobile phase was carried out according to the PRISMA system. The mobile phase optimization was carried out on TLC plates in unsaturated chambers. The resulting mobile phases were transposed to off-line, non-equilibrated OPLC and further to HPLC. The retention in TLC was measured at 37 selectivity points and in OPLC and HPLC at 13 points. Capacity factors (k′) and separation factors () were calculated in order to study the retention behaviour in the different systems. Two- and three-dimensional evaluations of k′ against selectivity points showed similar retention behaviours for the coumarins in TLC, OPLC and HPLC. The values for TLC, OPLC and HPLC showed similar patterns in the three-dimensional evaluations. The retention behaviour at different solvent strengths was also examined. According to quadratic regression, k′ showed a dependence on the change in solvent strength. OPLC, which can be considered as a “planar column” technique, and TLC are closely related methods, whereas HPLC shows a different behaviour in the elution process with regard to solvent strength.     

Separation and identification of some common isomeric plant triterpenoids by thin-layer chromatography and high-performance liquid chromatography

Chromatographic separation of 10 triterpenoids (α-amyrin, β-amyrin, δ-amyrin, lupeol, lupenon, lupeol acetate, cycloartenol, cycloartenol acetate, ursolic acid, oleanolic acid) and 2 sterols (stigmasterol and β-sitosterol) was studied. The chromatographic techniques included silica gel and reversed-phase (C₁₈ RP) thin-layer chromatography (TLC) and C₁₈ RP high-performance liquid chromatography (HPLC) using UV and mass spectrometric (MS) detection with atmospheric pressure chemical ionization (APCI). The TLC separation of the isomeric triterpenols lupeol, α-amyrin, β-amyrin and cycloartenol was achieved for the first time using C₁₈ RP-HPTLC plates. Cycloartenol could be separated from related compounds only on C₁₈ RP-TLC but not on the C₁₈ RP-HPLC. δ-Amyrin isolated from the tomato fruit surface extract could be separated from other amyrins only by HPLC. Tandem mass spectrometry allowed discrimination between the isomers lupeol, α-amyrin, β-amyrin, δ-amyrin, cycloartenol and between lupeol acetate and cycloartenol acetate. The combination of 3 TLC methods and 2 HPLC methods enables qualitative determination of all 12 compounds and proves to be useful for the analysis of plant extracts. It is recommended that TLC screening on silica gel and C₁₈ RP be performed before HPLC analysis.     

High-performance liquid chromatography and thin-layer chromatography for the simultaneous quantitation of rabeprazole and mosapride in pharmaceutical products

Simple, sensitive high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) methods are developed for the quantitative estimation of rabeprazole and mosapride in their combined pharmaceutical dosage forms. In HPLC, rabeprazole and mosapride are chromatographed using 0.01M 6.5 pH ammonium acetate buffer-methanol-acetonitrile (40:20:40, v/v, pH 5.70+/-0.02) as the mobile phase at a flow rate of 1.0 mL/min. In TLC, the mobile phase is ethyl acetate-methanol-benzene (2:0.5:2.5, v/v). Both the drugs are scanned at 276 nm. The retention times of rabeprazole and mosapride are found to be 4.93+/-0.01 and 9.79+/-0.02, respectively. The Rf values of rabeprazole and mosapride are found to be 0.42+/-0.02 and 0.61+/-0.02, respectively. The linearities of rabeprazole and mosapride are in the range of 400-2000 ng/mL and 300-1500 ng/mL, respectively, for HPLC; in TLC, the linearities of rabeprazole and mosapride are in the range of 400-1200 ng/spot and 300-900 ng/spot, respectively. The limit of detection is found to be 97.7 ng/mL for rabeprazole and 97.6 ng/mL for mosapride in HPLC; in TLC the limit of detection is found to be 132.29 ng/spot for rabeprazole and 98.25 ng/spot for mosapride. The proposed methods can be applied to the determination of rabeprazole and mosapride in combined pharmaceutical products.     

Preparative-scale chromatography of ecdysteroids of Serratula wolffli andrae

Numerous ecdysteroids are isolated from the herb of Serratula wolffii Andrae, a cultivated plant. The isolation procedure includes a variety of low-pressure liquid chromatography, thin-layer chromatography (TLC), gel chromatography, and high-performance liquid chromatography (HPLC) methods. The progress of separation is monitored by TLC, and the final proof of purity is carried out by HPLC. The isolation process involves the removal of proteins, flavonoids, chlorophylls, other sterines, etc. The purification also includes the separation of the target ecdysteroids from each other. Isolation of the pure compounds requires 2-8 chromatographic steps. The consecutive steps are based on the different physicochemical properties of the ecdysteroids. In some cases, a special peak-cut method employing a flush of dichloromethane into the dichloromethane-isopropanol-water mobile phase is used. This flush of dichloromethane leads to an almost perfect separation of otherwise unresolved peaks. Two ecdysteroids, 25-hydroxydacryhainansterone and 14-epi-20-hydroxyecdysone, are identified as natural products for the first time. The structure-chiroptical relationships for some ecdysteroids are also discussed.     

Oplopanphesides A-C, three new phenolic glycosides from the root barks of Oplopanax horridus

Three new phenolic glycosides, named oplopanphesides A-C (1-3), have been isolated from the root barks of Oplopanax horridus. Their structures were elucidated by a combination of spectroscopic analyses, including 1D- and 2D-NMR techniques. These phenolic glycosides possess a novel feature in their sugar moieties that a 3-hydroxy-3-methylglutaryl moiety was connected with C-6 of the β-D-glucopyranosyl group. Those compounds showed no cytotoxic effects against human cancer cell lines (MDA-231 and MCF-7) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.     

First Total Synthesis of Optically Active Oplopandiol Acetate, a Potent Antimycobacterial Polyyne Isolated from Oplopanax horridus

OPlopanax horridus, commonly known as devilal club, is a well-known shrub of westernNorth American forests. The inner bark and roots can be used for a variety of ailmentssuch as diabetes, rheumatism, tuberculosis, headache, and lung hemorrhage. OPlopandiolacetate l, a bioactive polyyne with significant anti-candida, antibacterial, and antimycobacterial activity, was isolated from O. horridus by Kobaisy in 1997. And itsabsolute configuration was determined to be (1 1 S, 16S) by the Mosher m…     

Determination of aflatoxins in food products by chromatography.

Several chromatographic methods for the determination of aflatoxins in agricultural and food products are reviewed. During the past two decades, identification and determination of aflatoxins were done by thin-layer chromatography (TLC) because it was easy, fast and inexpensive. However, high-performance liquid chromatography (HPLC) using fluorescence detection is now the method of choice for determining aflatoxins and is also growing in popularity for their identification. The reasons for selecting HPLC over TLC can be summarized as the ability to analyze for a wide variety of compounds, including compounds that are easily degraded by heat, light or air, the ease of adaptation to confirmatory procedures, the potential for automation and the dramatic improvement in instrumentation, including the development of increasingly sensitive fluorescence and electrochemical detectors and short, high-resolution, reversed-phase columns.     

Determination of quinidine in serum by spectrofluorometry, liquid chromatography and fluorescence scanning thin-layer chromatography

Quinidine is determined in serum by direct and extraction spectrofluorometry, by reflectance fluorescence scanning thin-layer chromatography (TLC), and by high-performance liquid chromatography (HPLC). Least-squares analyses of patients' sera (n = 62) analyzed first by direct fluorometry (x) and then HPLC (y) gave a slope of 0.52, an y-intercept of -0.40, a standard error of estimate of 0.65, and a correlation coefficient of 0.83. Comparison of patients' sera (n = 59) determined by extraction fluorometry (x) and then HPLC (y) gave a slope of 0.998, an y-intercept of -0.175, a standard error of estimate of 0.30, and a correlation coefficient of 0.96. Comparison of patients' sera (n = 36) by HPLC (x) and then reflectance fluorescence scanning TLC (y) gave a slope of 0.837, an y-intercept of 0.152, and a correlation coefficient of 0.94. Methaqualone and oxazepam interfere with HPLC. Within-run precision is 1.6, 1.0, 5.2 and 3.0% by direct fluorometry, extraction fluorometry, TLC and HPLC while between-run precision is 5, 3.5, 9 and 6.0%, respectively.     

Analysis of the Cinchona alkaloids by high-performance liquid chromatography and other separation techniques

The Cinchona alkaloids, which include the pharmaceuticals quinine and quinidine, continue to have a wide variety of important uses. A number of different chromatographic procedures have been developed for the qualitative and quantitative analysis of these compounds in a variety of sample matrices. Reversed-phase HPLC using ODS columns in combination with acidic mobile phases, and UV detection, is the most widely used method. Nevertheless, precautions need to be taken due to the strong silanophilic interactions which can occur with these analytes and the column surface, which can lead to poor peak shape and resolution. Different selectivity may be achieved in HPLC separations by use of alternative stationary phases, or by varying mobile phase pH. The specificity of detection systems may be improved by use of photodiode array UV detectors, or especially mass spectrometers. Thin-layer chromatography (TLC) provides a cheap alternative analytical method, which is especially useful for qualitative analysis. High-performance TLC, gas chromatography, capillary electrophoresis and capillary electrochromatography are all methods which after some development, could prove useful for Cinchona alkaloid separations.     

Analytical application of liquid adsorption chromatography of metal chelates

Summary A review of the most important achievements in the application of liquid adsorption chromatography (LAC) of metals as chelates is given. The advantages of the method in combination with an extraction preconcentration of metals are pointed out. The general problems of LAC of chelates (choice of a chelating reagent, requirements for chelates, conditions of formation and separation of complexes) are discussed. The applications of TLC and HPLC are considered separately. Special attention is paid to the analysis of specific objects. The analytical possibilities of TLC and HPLC are compared as applied to the separation and determination of metal chelates. The fields of application and the perspectives of developing the methods in inorganic analysis are estimated.

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Analytische Anwendung der Flüssig-Adsorptions-Chromatographie von Metallchelaten

     

Reduction of silanophilic interactions in liquid chromatography with the use of ionic liquids

A suppression of silanophilic interactions by the selected ionic liquids added to the mobile phase in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) is reported. Acetonitrile was used as the eluent, alone or with various concentrations of water and phosphoric buffer pH 3. Selectivity of the normal (NP) and the reversed (RP) stationary phase material was examined using a series of proton-acceptor basic drugs analytes. The ionic liquids studied appeared to significantly affect analyte retention in NP-TLC, RP-TLC and RP-HPLC systems tested. Consequently, the increased separation selectivity was attained. Due to ionic liquid additives to eluent even analytes could be chromatographed, which were not eluted from the silica-based stationary phase materials with 100% of acetonitrile in the mobile phase. Addition of ionic liquid already in very small concentration (0.5%, v/v) could reduce the amount of acetonitrile used during the optimization of basic analytes separations in TLC and HPLC systems. Moreover, the influence of temperature on the separation of basic analytes was demonstrated and considered in practical HPLC method development.     

Isolation of experimental anti-AIDS glycerophospholipids by micro-preparative reversed-phase high-performance liquid chromatography.

The experimental anti-AIDS glycerophosphatidic acid: nucleoside (sn-1/sn-2 diacylglycerol:dideoxynucleotide) drugs 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) and 2',3'-dideoxycytidine monophosphate diglyceride (ddC-MP-DG) were isolated and purified by reversed-phase high-performance liquid chromatography (HPLC). The chromatographic separation was based on the glycerophospholipid moiety of the drugs and detection of the nucleoside component. The separations were optimized on method development columns packed with the stationary phase to be used in the micro-preparative column and monitored by a UV detector. Fractions were collected and analyzed for purity by analytical-scale HPLC and by thin-layer chromatography (TLC). The purity of the recovered drugs based on UV and light-scattering detection and on TLC was greater than 99%. The purified compounds were isolated for studies on structure confirmation, physical, biophysical and formulation properties and anti-HIV efficacy in culture.     

The use of thin-layer chromatography in the assessment of the quality of lutein-containing dietary supplements

Bivariant multiple development thin-layer chromatography technique (BMD–TLC) along with high-performance liquid chromatography–diode array detection–electrospray ionization–mass spectrometry (HPLC–DAD–ESI–MS) analysis was used in determination of lutein or lutein mixed with zeaxanthin in eight dietary supplements. The developed two-step TLC separation procedure combined purification, compaction of samples and separation of the analyzed compounds what significantly shortened and simplified samples preparation. Qualitative analysis was based on co-chromatography with reference substances and HPLC–DAD–ESI–MS analysis. It was revealed that three of eight dietary supplements did not contain lutein. In turn, quantitative analysis with the use of developed TLC conditions along with densitometry showed that the amount of lutein or its mixture with zeaxanthin in the others differed from that claimed by producers.

     

Chromatographic methods as tools in the field of mycotoxins

Achievements in the applications of chromatographic techniques in mycotoxicology are reviewed. Historically, column chromatography (CC) and paper chromatography (PC) were applied first, followed by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and gas chromatography (GC). Although PC techniques are no longer used in the analysis of mycotoxins, selected applications of PC are included to underline historical continuity. The most important achievements published from 1980 onwards are described. They include clean-up methods, TLC, CC, HPLC and GC of mycotoxins in environmental samples, foods, feeds, body fluids and in studies on biosynthesis and biotransformations of mycotoxins. Advantages and disadvantages of chromatographic techniques used in mycotoxicology are also evaluated.     

Determination of 8-hydroxyquinoline sulfate in tuberculin solutions by planar and high performance liquid chromatography

Summary TLC and HPLC methods for the determination of the preservative, 8-hydroxyquinoline sulfate in PPD-T tuberculin solution were developed. The planar chromatography method involved separation of 8-hydroxyquinoline sulfate on a TLC plate using a butyl-acetate: formic acid: 2-propanol mobile phase, detection and quantitation by densitometric scanning. The HPLC method was on a LiChrosorb RP-18 column with acetonitrile-water (65:35 v/v) mobile phase, adjusted to pH 3.05 by phosphoric acid. Linearity, reproducibility and accuracy were found to be satisfactory. Under selected conditions, the limit of detection (LOD) of both methods was similar-about 25 ng. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Thin-layer chromatography and multivariate data analysis of willow bark extracts

In most cases the pharmacological activity of plant extracts is not assigned to single components and often not all active ingredients are known. Approaches other than those considering single compounds only to analyze plant material have proven helpful for a better characterization of extracts in their entirety. In this study extracts of willow bark are analyzed by high-performance thin-layer chromatography (HPTLC) and two different pharmacological tests [the 2,2'-azobis (2-amidinopropane) dihydrochloride reaction and the xanthine/xanthine oxidase reaction] with the help of multivariate data analysis. Described are two models using the results of the chromatographic study of 22 various extracts of willow bark and their pharmacological properties. The chromatographic data are obtained by a special TLC scanner that enables measurement of HPTLC tracks simultaneously in the range of lambda = 200-400 nm. Additionally, the developed models are used to predict the activity of another three extracts of willow bark demonstrating the quality of the model.     

Separation of peptides on Empore thin-layer chromatography sheets and blotting onto polyvinylidene difluoride membranes with subsequent gas phase sequencing.

Methods for the separation of peptides on a new type of thin-layer chromatography (TLC) sheet and blotting onto polyvinylidene difluoride (PVDF) membranes with subsequent gas phase sequencing are described. For validation, the A and B chain of insulin were chromatographed on Empore TLC sheets and either extracted or blotted onto PVDF membranes. The advantages and disadvantages of thin-layer chromatography on Empore sheets versus high performance liquid chromatography (HPLC) are discussed, along with the possibility of combining the two methods. In addition, TLC was combined with electrophoresis (fingerprinting) for the separation of complex peptide mixtures. Blotting from TLC sheets onto PVDF membranes was performed in two ways: contact diffusion and electrophoretic transfer. In our experiments electroblotting was more effective. Amino acid sequence determination of the B chain of insulin was possible both after extraction from the TLC sheet and after blotting onto PVDF membranes. In the former case, liquid phase sequencing and, in the latter case, gas phase sequencing was performed. The possibility to blot from TLC sheets onto membranes, e.g. PVDF, may prove useful in many fields, for example in biochemistry, and in molecular and cell biology.     

Chromatography of Trichothecene Mycotoxins

Abstract

Trichothecene mycotoxins occur in agricultural commodities and can cause problems from feed refusal to death in animals. This paper describes chromatographic methods for selective analysis for trichothecene mycotoxins. These methods include gas chromatography (GC), thin layer chromatography (TLC), and high pressure liquid chromatography (HPLC). The trichothecene analysis methods by GC and TLC are shown to have a greater sensitivity than in HPLC for the underivatized mycotoxins.     

Antimicrobial and cytotoxic activities of leaves, twigs and stem bark of Scutia buxifolia Reissek

The antimicrobial and cytotoxic activities of the dichloromethane, ethyl acetate and butanolic fractions from the leaves, twigs and stem bark of Scutia buxifolia were evaluated using the broth microdilution method and the brine shrimp lethality method, respectively. Phytochemical analysis was performed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The antimicrobial results demonstrated that the strongest effect occurred with the butanol fraction from the twigs and the ethyl acetate fraction from the stem bark against Saccharomyces cerevisiae (minimal inhibitory concentration; MIC?=?62.5?μg?mL(-1)), whereas the ethyl acetate and butanolic fractions from the twigs and stem bark were effective against Pseudomonas aeruginosa and Staphylococcus aureus, with MIC values ranging from 125 to 500?μg?mL(-1). LD(50) values varied from 50.00?±?0.22 to 82.23?±?0.34?μg?mL(-1). Quercetin, quercitrin, isoquercitrin and rutin were identified by HPLC and may be partially responsible for the antimicrobial activities observed. This study reports for the first time the antimicrobial and cytotoxic activities of S. buxifolia leaves, twigs and stem bark.