Performance Thin Layer Chromatography Research Articles

Stability-indicating reversed-phase high-performance thin-layer chromatography/densitometry estimation of lafutidine in bulk and tablets

Lafutidine (LTD) is a histamine H2 receptor antagonist, with anti-ulcer and mucosal protective activity. A sensitive and selective stability-indicating reversed- phase high -performance thin-layer chromatography (RP-HPTLC)/Densitometry method is suggested for the determination of LTD in the presence of its acid, alkaline, oxidative and dry heat degradation products. Stress testing of LTD was done according to the International Conference on Harmonization (ICH) guidelines in order to validate the stability-indicating power of the analytical procedures. Stress testing demonstrated that LTD underwent acid, alkaline, oxidative and dry heat degradation; on the other hand, it showed stability towards neutral and photo degradation. Chromatography was performed on aluminum-backed silica gel 60 RP-18 F254 S plates with Methanol: Water: Triethylamine 8: 2: 0.5 (v/v) as mobile phase. Densitometric scanning was performed at 276 nm. The system gave compact bands for LTD (Rf 0.67 ? 0.02). Linear regression analysis data for the calibration plots discovered a good linear relationship with r2 > 0.99 in the working concentration range 500 to 3000 ng per band. The limits of detection and quantification were 23.51 ng and 71.26 ng, respectively. The developed method can routinely be used for analysis of LTD in bulk and pharmaceutical formulations.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

Indigenous anti-ulcer activity of Musa sapientum on peptic ulcer.

Background:Peptic ulcer disease (PUD), encompassing gastric and duodenal ulcers is the most prevalent gastrointestinal disorder. The pathophysiology of PUD involves an imbalance between offensive factors like acid, pepsin and defensive factors like nitric oxide and growth factors. The clinical evaluation of antiulcer drugs showed tolerance, incidence of relapses and side-effects that make their efficacy arguable. An indigenous drug like Musa sapientum possessing fewer side-effects is the major thrust area of present day research, aiming at a better and safer approach for the management of PUD.Material and Methods:The unripe plantain bananas (Musa sapientum) were shade-dried, powdered and used for phytochemical analysis and as antiulcer drug. In our present study Group I rats served as control and were treated with saline, Group II was indomethacin-induced ulcerated rats, Group III received aqueous extract of Musa sapientum along with indomethacin and Group IV received esomeprazole along with indomethacin for 21 days. The anti-ulcerogenic activity was investigated by performing hematological, mucosal, antioxidant profile in comparison with the standard drug esomeprazole.Results:Our findings from High - Performance Thin Layer Chromatography (HPTLC) analysis showed that Musa sapientum has an active compound a monomeric flavonoid (leucocyanidin) with anti-ulcerogenic activity. Results were expressed as mean ± SD. All our results are in congruous with the results of standard drug esomeprazole.Conclusion:It could be clearly concluded that administration of the aqueous extract of Musa sapientum at the dose used in this study tends to ameliorate ulcers. Its use in indigenous medicine should be scientifically scrutinized with further research.

Ganglioside GM3 is stably associated to tyrosine-phosphorylated ErbB2/EGFR receptor complexes and EGFR monomers, but not to ErbB2

Gangliosides are known to modulate the activation of receptor tyrosine-kinases (RTKs). Recently, we demonstrated the functional relationship between ErbB2 and ganglioside GM3 in HC11 epithelial cell line. In the present study we investigated, in the same cells, the ErbB2 activation state and its tendency to form stable molecular complexes with the epidermal growth factor receptor (EGFR) and with ganglioside GM3 upon EGF stimulation. Results from co-immunoprecipitation experiments and western blot analyses indicate that tyrosine-phosphorylated ErbB2 and EGFR monomers and stable ErbB2/EGFR high molecular complexes (heterodimers) are formed following EGF stimulation, even if the receptors co-immunoprecipitates also in the absence of the ligand; these data suggest the existence of pre-dimerization inactive receptor clusters on the cell surface. High performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of the ganglioside fractions extracted from the immunoprecipitates demonstrate that GM3, but not other gangliosides, is tightly associated to the tyrosine-phosphorylated receptors. Furthermore, we show that GM3 is preferentially and in a SDS-resistant manner associated to the activated ErbB2/EGFR complexes and EGFR monomer, but not to ErbB2. Altogether our data support the hypothesis that the modulating effects produced by GM3 on ErbB2 activation are mediated by EGFR.

High Capacity Homogeneous Non-Radioactive Cortisol Detection Assays for Human 11β-Hydroxysteroid Dehydrogenase Type 1

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.

BIOCHEMICAL CHARACTERIZATION OF INHIBIN HORMONE IN THE OVARY OF SHE CAMEL (CAMELUS DROMEDARIES)

Isolation, purification and advanced characterization of hormone inhibin in ovary of female camel are aimed in this study. Pooled follicular fluid was collected from the ovaries of she-camel irrespective to physiological status and age of the animals. Follicular fluid was subjected to 2 types of gel filtration chromatography; Sephacryl S-200 where three peaks of proteins was obtained. The suspected peak to contain inhibin (peak III) resolved from S-200 was subjected to Sephadex G-100 where three peaks were obtained, third peak was suspected to contain inhibin in pure form. To verify the purity of the isolated hormone, the lyophilized fraction containing inhibin was subjected to analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under both non reducing condition, stained with silver nitrate where a single band was resolved, and under reducing condition, where five bands resolved (ranging between 58.8 to 32.3 KDa). Using Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) to fractionate the third peak, subunits of inhibin hormone eluted at different retention times. High Performance-Thin Layer Chromatography (HP-TLC) used to determine the N-terminal amino acids contents of the third peak and 7 amino acids resolved with different concentrations where histidine was the most abundant of the amino acids.

Chiral Separations of Ibuprofen and Propranolol by TLC. A Study of the Mechanism and Thermodynamics of Retention

In contrast with gas and column liquid chromatography, both of which enable very efficient separation of enantiomers, thin‐layer chromatography (TLC) has never proved particularly successful in the same field. This can be regarded as puzzling because although the performance of TLC is substantially lower than that of the instrumental modes of chromatography, it still seems efficient enough to ensure even difficult separations of pairs of analytes. There is a steady demand for simple, inexpensive, and successful chiral separations, preferably executed with the aid of TLC. The best proof of this is a few documented and promising attempts reported by reliable laboratories in developing countries. Some of these reports, however, describe experiments performed with glass plates coated in the laboratory, which gives rise to questions regarding the accuracy and repeatability of the results obtained. Similar concerns are evoked by traditional visualization of the outcome of a separation by use of dyeing agents, rather than by densitometry (which furnishes concentration profiles of the bands and the possibility of in‐situ identification also). In this study, we have repeated chiral separations of two widely used drugs, ibuprofen and propranolol, adapting working conditions reported elsewhere to a system based on standardized and commercially available chromatographic plates. We also performed detection and identification on the developed chromatograms by means of densitometry. In our experiments with the modified chromatographic procedures, the results obtained proved at least as good as those reported in the original papers and occasionally somewhat better. For both ibuprofen and propranolol, preliminary thermodynamic evaluation of the standard chemical potentials of adsorption (Δμa) for each of the two enantiomers considered was also performed. The results obtained look promising and realistic; it seems that adsorption TLC can be used in the future for thermodynamic assessment of the racemization process.

The presence of orellanine in spores and basidiocarp from Cortinarius orellanus and Cortinarius rubellus

This is the first report quantifying the orellanine content in basidiospores. The toxin content and tissue distribution of orellanine were determined from Cortinarius orellanus (Fr.) Fr. and Cortinarius rubellus Cooke. Basidiospores, the basidiocarp, divided into cap and stem, and mycorrhiza roots were analyzed to determine the amount of orellanine by reversed phase high performance liquid chromatography and thin layer chromatography. The orellanine contents in spores were 0.31% (C. orellanus) and 0.09% (C. rubellus). In caps, we found the toxin content to be 0.94% (C. orellanus) and 0.78% (C. rubellus), in stems 0.48% (C. orellanus) and 0.42% (C. rubellus) and in mycorrhiza roots from C. rubellus we determined the orellanine contents to 0.03%. In addition, extracts from the different structures of the basidiocarp of C. orellanus and C. rubellus, with an orellanine content corresponding to 25 nmol, inhibited the growth of Bacillus subtilis.

Visual Estimation of Aflatoxin Production in Peanut with Aspergillus Norsolorinic Acid Mutants1

Abstract Peanut seed and pods are susceptible to contamination by aflatoxin (AF), a carcinogenic mycotoxin produced by Aspergillus flavus Links Fr. and A. parasiticus Speare. Efforts to evaluate peanut lines for resistance to AF contamination have been impeded by limitations to the methodologies available for AF detection. AF cannot be seen by visible light and its detection involves grinding seed tissue in organic solvents, separation of phases, and detection by ELISA, high performance liquid chromatography (HPLC) or thin layer chromatography. These methodologies are time-consuming, expensive, labor-intensive, and are uninformative in defining the tissues of the peanut seed and pod that are most frequently contaminated with AF. Aspergillus AF mutants which accumulate norsolorinic acid (NOR), an orange-pigmented AF pathway intermediate, provide an easy and convenient mean to detect AF contamination. A visual rating scheme for NOR contamination of peanut seed was developed that correlated favorably to HPLC detection of both NOR and AF (r = 0.96 and 0.95, respectively). When screening the 38 plant progenies that comprise Tamspan 90 (a spanish cultivar), NOR was first seen in the intercotyledonary cavity and the interfacial surface of cotyledons and testae in seeds examined from infected pods. Immature pods were often heavily contaminated with NOR. Six of the 38 lines accumulated low levels of NOR in two laboratory tests. Additional studies are needed to determine if these results are predictive of aflatoxin contamination under field conditions.

Toxin analysis of two low toxicity species of dinoflagellateAlexandrium

Abstract The content of paralytic shellfish poisons (PSP) in cultured cells of the dinoflagellate Alexandrium lusitanicum and of natural phytoplankton blooms of Alexandrium tamarense, were analyzed by high performance liquid chromatography and thin layer chromatography. The extracts of both species killed mice without showing clearly any of the typical signs for PSP. In the HFLC and TLC analysis no recognized toxins were detected in either of the two species.

Antibodies against platelet glycosphingolipids: detection in serum by quantitative HPTLC-autoradiography and association with autoimmune and alloimmune processes

In order to assess the importance of glycosphingolipids (GSL) in the immunology of the platelet, serum antibody binding to immobilized, purified platelet GSLs have been quantitatively measured via high- performance thin-layer chromatography (HPTLC), 125I-radio- immunolabeling, autoradiography, and densitometry. Thirteen neutral GSL bands were detected at Rf.03 through .64 (CHCl3-CH3OH-H2O, 65:25:4) after extraction and chromatography (DEAE-Sephadex and Bio-sil A). Both IgM and IgG serum antibody binding was determined for 50 subjects from four groups: normal blood donors (NBD, n = 18); leukemia patients with nonimmune thrombocytopenia (NIT, n = 10); patients with systemic lupus erythematosus (SLE, n = 10); and patients with chronic autoimmune thrombocytopenia (CATP, n = 12). The ABO typing of these 50 subjects also allowed correlation of serum antibody binding with A blood group antigen expression. These studies reveal: (1) anti-GSL binding at band .06 is associated with blood group A alloantigen expression for both IgG and IgM (P less than .0001) antibodies; (2) binding at bands .17, .27, and/or .46 is associated with general autoimmunity (SLE and CATP) for IgM (P less than .0001); (3) binding at bands .35 and/or .38 is associated with platelet-specific autoimmunity (CATP) for IgG and/or IgM (P less than .005); and (4) binding at bands .03, .20, .23, and/or .43 is frequently observed for sera from all groups. The platelet specificity of bands .35 and .38 was confirmed by comparative studies with human intestinal smooth muscle GSLs. Quantitation of the intensity of CATP-associated anti-GSL binding (86 +/- 88 mm2) is comparable to anti-A alloantigen binding (57 +/- 52 mm2). Two of the GSL bands associated with SLE and CATP appear to be the long-chain fatty-acyl forms of globotriaosyl ceramide (.27) and globotetraosyl ceramide (.17), which are the Pk and P blood group antigens, respectively. These studies indicate that neutral GSLs may be important antigens in both autoimmune and alloimmune processes involving the blood platelet.

A non-instrumental, qualitative test for free gold in geological samples

A relatively unsophisticated method for the qualitative identification of gold in geological samples is described. This method uses a combination of aqua regia digestion, ethyl ether extraction, solution preconcentration, separation by high performance-thin layer chromatography, and flame reduction visualization to achieve selective detection of free gold at concentrations down to 0.09 μg Au g −1 sample. The analytical results obtained with this non-instrumental method demonstrate the feasibility of an inexpensive “gold test kit” for preliminary exploration surveys.

Characterization of the phospholipid and fatty acid composition of Sendai virus.

The lipid composition of Sendai virus, propagated in chicken eggs, was analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas-liquid chromatography (GLC). Phosphatidylcholine was found to be the dominant phospholipid (37.3%) with phosphatidylethanolamine (26.8%) and phosphatidylserine (12.0%) also present in significant amounts. Analysis of the fatty acid methyl esters revealed that the dominant fatty acids in total phospholipid were: C16:0 (17.6%), C18:0 (15.4%), C18:1 (n-9) (22.0%), and C24:0 (6.0%). Cardiolipin, phosphatidylserine, and sphingomyelin contained higher levels of saturated fatty acids relative to phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.

Analysis of N3,N10-methenyltetrahydrofolic acid by high performance liquid and thin layer chromatography

Analysis of N3,N10-methenyltetrahydrofolic acid by high performance liquid and thin layer chromatography

Fluidity properties of isolated chloroplast thylakoid lipids

Chloroplast thylakoid lipids have been isolated free of photosynthetic pigments using a combination of high performance liquid and thin layer chromatography. The hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into aqueous dispersions of the isolated lipids in order to investigate dynamic and structural properties of the resulting bilayer membranes. Time dependent fluorescence anisotropy decays have been measured and analysed assuming the wobbling-in-cone model (Kinosita et al., Biophys J 20 (1977) 289-305). The DPH fluorescence lifetimes and the static and dynamic fluorescence anisotropy decay parameters for the probe in a total lipid mixture or in pure digalactosyldiacylglycerol (DGDG), changed in a predictable way with increasing temperature (10°-36°C). For a given temperature, it was found that the total lipid mixture was in general less ordered and showed greater dynamic motion as judged from DPH fluorescence anisotropy and compared with the pure DGDG system, although at 36°C differences in dynamic parameters were less evident. Overall the results obtained emphasize the highly fluid nature of thylakoid membrane lipids and give a basis for investigating how intrinsic proteins modify structural and dynamic properties of the in vivo membrane.

Identification and Estimation of the Alkaloids of Rauwolfia serpentina by High Performance Liquid Chromatography and Thin Layer Chromatography

Abstract A procedure is presented for the identification and estimation of some of the alkaloids of Rauwolfia serpentina by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Two extraction procedures were studied, one essentially similar to the current AOAC method, and one using warm methanol. For HPLC, a μPorasil column, methanol as eluting solvent, and a fluorometric delector were used. Rescinnamine was detected at 330 nm, at which wavelength reserpine fluorescence is negligible. Reserpine was detected at 280 nm, where rescinnamine fluorescence is small. Other alkaloids detected were raubasinine, ajmalicine, yohimbine, ajmaiine, and serpentine. For TLC, CHCI3-CH3OH 197+ 3) and CHCI3-CH3OH (80 + 20) were used as developing solvents and spots were detected under long- and shortwave UV light. A semiquantitative IICprocedure was also developed for serpentine, the content of which was found to be in the 0.2-0.25% range.

Detection of Adulteration in Blackberry Juice Concentrates and Wines

Adulteration of blackberry juice concentrates and wines with juice of sorbitol-containing fruits was detected by determining carbohydrates by high performance liquid chromatography, gas-liquid chromatography, and thin layer chromatography. Sorbitol is not fermented by yeast and can be detected in wines made from blackberry juice concentrates that contain sorbitol. High levels of sorbitol and quinic and malic acids suggest that an imported blackberry concentrate may have been adulterated with plum. Degradation of anthocyanin pigments during processing and storage limits the utility of pigment analyses in detecting adulteration.

Analysis of Coumarone-indene Resin Coated on Citrus Fruits

Methods were developed for the detection and determination of coumarone-indene (CI) resin coated on citrus fruits.High performance thin-layer chromatography (HPTLC) was utilized to separate CI resin effectively; the first development was carried out with benzene, and the second with tetrachloroethylene. Five distinct spots were observed under UV (254nm) irradiation.Chromogenic agents such as concentrated sulfuric acid and antimony trichloride were also investigated to achieve the specific and sensitive identification of CI resin, and a satisfactory result was obtained.Quantitative determination of CI resin was carried out by pyrolysis gas chromatography (FID); by using indene of the main pyrolysis product as an index, it was possible to determine 5ppm CI resin.The recovery of CI resin coated on citrus fruits was investigated and found to be more than 95%.