High-performance Thin-layer Plates Research Articles

Equol determination in cattle manure by HPTLC-DART-TOF-MS

We present a planar chromatographic separation method for the phytoestrogenic active compound equol, separated on silica gel High Performance Thin Layer-plates (HPTLC-plates, Merck, 1.00934). It could be shown that an ethanolic cattle manure extract contains this phytoestrogenic active compound to a larger amount. As solvents for the mobile phase have been used methyl t-butyl ether and cyclohexane (1 + 1, v/v). After separation the plate was scanned with a very specific working TOF-DART-MS system (Time Of Flight – Direct Analysis in Real Time – Mass Spectrometry) using the (M + 1)+-signal of equol. The detection in combination with an HPTLC-separation allows very specific detecting of equol in cattle manure, although that is a very challenging matrix. Preliminary results show that the content of equol in liquid manure is about 70 µg/g.

SEPARATION OF FOUR CATIONIC SURFACTANTS ON SILICA GEL 60 F254 HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHIC PLATES

The chromatography of four cationic surfactants was performed on silica high performance thin-layer plates with mixed solvent system of methanol and ethylenediaminetetraacetic acid (EDTA). The resolution of a mixture of Benzyltrimethylammonium chloride (BTAC), Dodecyltrimethylammonium chloride (DTAC), Tetrabutylammonium bromide (TBAB), and Methyltrioctylammonium bromide (MTOB) was achieved by using methanol:5% aqueous EDTA (7:3,v/v) as a solvent system. The effect of concentration of EDTA on the mobility of all the four cationic surfactants was examined. The limit of detection of BTAC, DTAC, TBAB, and MTOB estimated was 0.65, 0.15, 0.15, and 0.31 µg/zone, respectively. The developed method was utilized to identify these surfactants in different spiked water samples after their preliminary separation.

Identification and separation of lead (II), nickel (II), and cobalt (II) on silica gel 60 F254high-performance thin-layer chromatographic plates with mixed aqueous sodium dodecyl sulfate-oxalic acid solvent system

The chromatographic separation of three metal cations was performed on high-performance thin-layer plates (silica gel 60F254) with mixed sodium dodecyl sulphate (SDS) and oxalic acid. The effect of concentration of surfactant and additive effect of different carboxylic acids on the mobility of metal ions were investigated. The resolution of a mixture of Pb2+, Ni2+, and Co2+ was achieved by using 0.2 M SDS plus 0.08 M oxalic acid, 1:9, v/v as a solvent system. The developed method was utilized to identify these metal ions in different spiked water samples after their preliminary separation.

Fatty Acid Transport Protein 4 Is the Principal Very Long Chain Fatty Acyl-CoA Synthetase in Skin Fibroblasts

Fatty acid transport protein 4 (FATP4) is a fatty acyl-CoA synthetase that preferentially activates very long chain fatty acid substrates, such as C24:0, to their CoA derivatives. To gain better insight into the physiological functions of FATP4, we established dermal fibroblast cell lines from FATP4-deficient wrinkle-free mice and wild type (w.t.) mice. FATP4 -/- fibroblasts had no detectable FATP4 protein by Western blot. Compared with w.t. fibroblasts, cells lacking FATP4 had an 83% decrease in C24:0 activation. Peroxisomal degradation of C24:0 was reduced by 58%, and rates of C24:0 incorporation into major phospholipid species (54-64% decrease), triacylglycerol (64% decrease), and cholesterol esters (58% decrease) were significantly diminished. Because these lipid metabolic processes take place in different subcellular organelles, we used immunofluorescence and Western blotting of subcellular fractions to investigate the distribution of FATP4 protein and measured enzyme activity in fractions from w.t. and FATP4 -/- fibroblasts. FATP4 protein and acyl-CoA synthetase activity localized to multiple organelles, including mitochondria, peroxisomes, endoplasmic reticulum, and the mitochondria-associated membrane fraction. We conclude that in murine skin fibroblasts, FATP4 is the major enzyme producing very long chain fatty acid-CoA for lipid metabolic pathways. Although FATP4 deficiency primarily affected very long chain fatty acid metabolism, mutant fibroblasts also showed reduced uptake of a fluorescent long chain fatty acid and reduced levels of long chain polyunsaturated fatty acids. FATP4-deficient cells also contained abnormal neutral lipid droplets. These additional defects indicate that metabolic abnormalities in these cells are not limited to very long chain fatty acids.

Ceramide 1-Phosphate, a Mediator of Phagocytosis

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.

A New Anionic Micellar Mobile Phase System for the Normal-Phase Thin-Layer Chromatography of Amino Compounds: Simultaneous Separation of Aminophenol Isomers

The chromatography of some amino compounds was performed on high-performance thin-layer plates (silica-gel 60 F254, catalog no. 1.05554, Merck, Germany) using aqueous and alcoholic (MeOH) and aqueous–organic solutions of cationic, anionic, and nonionic surfactants. The results obtained with 0.01 M aqueous sodium dodecyl sulfate (SDS) have been compared with distilled water (i.e., zero SDS) as the eluent. The effects of the surfactant concentration, the nature of the alcohol, and the presence of inorganic salts (NaCl, LiCl, CaCl2, and CoCl2) in the mobile phase were examined in order to understand the mobility pattern of amino compounds. Among the added salts, CaCl2 was found to be the most effective for facilitating an analytically important separation of coexisting ortho-, meta-, and para-aminophenols. The TLC system consisting of a precoated high-performance silica layer as the stationary phase and 0.01 M methanolic SDS plus 0.1 M CaCl2 in a 3 : 7 ratio as the mobile phase was identified as the most suitable system for the separation of o-, m-, and p-APHS from their mixtures. The influence of various impurities, such as amines, phenols, and inorganic cations, on the mobility and the separation of coexisting aminophenol isomers has also been examined. The lower limits of detection of aminophenols were determined on HPTLC plates using I2 vapors as the detection reagent.

P.1.043 - Toxicity of antidepressant drugs: A comparative in vitro study

An immunostaining procedure has been developed for the assay of the gangliotetraose gangliosides and sulfatide in cerebrospinal fluid. Gangliosides of the gangliotetraose series were individually determined with cholera toxin B-subunit (CT-B) and an anti CT-B monoclonal antibody after chromatography and sialidase hydrolysis to GM1 on high performance thin-layer plates. Sulfatide was determined by thin-layer chromatography using an anti-sulfatide antibody. The method was applied to normal cerebrospinal fluid from 20 adults and 30 children. The concentration of the gangliotetraose series gangliosides in adults varied from 100–300 nmol/l with a mean value of 230 ± 56 nmol/l. Corresponding values for sulfatide were 30–225 nmol/l and 140 ± 46 nmol/l. The values for gangliosides and sulfatide in children increased during development. The major gangliosides in cerebrospinal fluid of adults were GTlb and GDlb and in children GDla and GTlb.

Characterization of gangliosides by two-dimensional high-performance thin-layer chromatography

A method for the qualitative and quantitative analysis of complex mixtures of gangliosides by computer-assisted image densitometry is reported. Gangliosides were applied to silica gel high-performance thin-layer plates and developed in two dimensions using one of several solvent systems. The plates were developed with a resorcinol reagent and the gangliosides were visualized as bluish-purple bands or spots. The plates were scanned with a Biolmage Visage 110 image analyzer and saved to disk. The densities of the spots were determined and the quantities of gangliosides determined by comparison with an internal standard, GM 4. Two-dimensional chromatography using chloroform-methanol-0.2% CaC1 2 (50:40:10) in the first dimension and n-propanol-ammonia(aq.) (6:3) in the second dimension gave linear and reproducible results when analyzed by image densitometry. Gangliosides isolated from human plasma were resolved with the above method. We suggest that the use of image densitometry improves the accuracy of quantitative analyses of complex mixtures of gangliosides by thin-layer chromatography.

Determination of gangliosides and sulfatide in human cerebrospinal fluid with a microimmunoaffinity technique

An immunostaining procedure has been developed for the assay of the gangliotetraose gangliosides and sulfatide in cerebrospinal fluid. Gangliosides of the gangliotetraose series were individually determined with cholera toxin B-subunit (CT-B) and an anti CT-B monoclonal antibody after chromatography and sialidase hydrolysis to GM1 on high performance thin-layer plates. Sulfatide was determined by thin-layer chromatography using an anti-sulfatide antibody. The method was applied to normal cerebrospinal fluid from 20 adults and 30 children. The concentration of the gangliotetraose series gangliosides in adults varied from 100–300 nmol/l with a mean value of 230 ± 56 nmol/l. Corresponding values for sulfatide were 30–225 nmol/l and 140 ± 46 nmol/l. The values for gangliosides and sulfatide in children increased during development. The major gangliosides in cerebrospinal fluid of adults were GTlb and GDlb and in children GDla and GTlb.

Laser microprobe mass spectrometry of selected compounds directly from normal phase high-performance thin-layer plates

Direct analysis of high-performance thin-layer chromatography (HPTLC) plates by laser microprobe mass spectrometry has been demonstrated. A series of general organics and ionic surfactants were examined as reference materials and after appalication to normal-phase HPTLC plates. Spectra were generated, containing structurally significant ions and a contribution from the silica chromatography medium. After chromatography in a solvent system, spectra were weak but included sufficient compound-specific ions to permit structural confirmation. The identification of total unknowns by this means alone would at present be speculative but, supplemented by visualisation reagents and known chromatographic reference points, valuable information can be gained.

Occurrence of an endogenous inhibitor of platelet-activating factor in rat liver.

Hepatocytes obtained from livers derived from fed rats perfused with a collagenase-containing mixture were found to contain significant levels of platelet-activating factor activity as isolated by Silica Gel G thin layer chromatography. However, when soybean trypsin inhibitor was included in the collagenase-containing perfusion medium for hepatocyte preparation, platelet-activating factor activity could not be detected on Silica Gel G chromatograms. Examination of the lipids extracted from freeze clamped perfused rat livers revealed low, but detectable, levels of platelet-activating factor. Further investigation of these observations indicated that a lipid-like inhibitor was present in freeze-clamped perfused livers as well as in hepatocytes isolated in the presence of soybean trypsin inhibitor. In each instance platelet-activating factor and this newly discovered inhibitor, which comigrated at the same RF value on Silica Gel G thin layer chromatography plates, could be separated by further chromatography on high performance thin layer plates. The present study shows that platelet-activating factor is present in unstimulated liver and that its detection is masked by an endogenous lipid-like inhibitor.

Mass spectrometric identification of the pentasialoganglioside GP1c of embryonic chicken brain.

Gangliosides were extracted from 11-day-old chicken embryos and finally purified by chromatography on high performance thin-layer plates. Four fractions migrating more slowly than ganglioside GQ1b were obtained by preparative thin-layer chromatography. With the aid of negative ion fast atom bombardment mass spectrometry, one of these could be identified as GP1c.

Reversed-phase high-performance thin-layer chromatography of mebeverine hydrochlordie and related compounds

The chromatographic behaviour of mebeverine hydrochloride, a musculotropic spasmolytic agent, and some related compounds, i.e. degradation compounds and intermediates from the synthesis, on reversed-phase pre-coated high-performance thin-layer plates is described. The retention and selectivity on different commercially available layers are studied in connection with different eluent properties. The developed method is applied to the quantitative determination of mebevirine hydrochloride. Excellent selectivity and good reproducibility are obtained, and the method provides a combined test of assay, purity and stability.

Solid-phase biosynthesis on high performance thin-layer plates of blood group glycosphingolipids II

A new method for the study of carbohydrate chain biosynthesis of amphipathic glycosphingolipids is presented. The method takes advantage of the thin-layer plate which is used as a solid matrix of precursor glycosphingolipids in biosynthetic experiments after chromatographic development of the precursor sample. The method is shown to be simple, fast and sensitive. It minimizes the risk of adding unwanted exogenous precursors and abolishes the need for tedious purification of products after incubation. The method opens up new possibilities for the biosynthetic study of mixtures of glycosphingolipids.

A modified high-performance thin-layer plate for the separation of purines and pyrimidines

Several ways of using the recently developed high-performance thin-layer chromatography (HPTLC) precoated plate NH 2 F 254 s to separate purines and pyrimidines are described. This precoated plate is coated with silica gel 60 which has been chemically modified with alkylamino groups. In view of the chemical properties of the functional groups bonded to the silica gel matrix, the HPTLC precoated plate NH 2 F 254 s can be considered to be a weak basic ion-exchange plate. In aqueous eluants the substances are separated principally according to charge differences. The HPTLC precoated plate NH 2 F 254 s can, however, also be used to separate uncharged, polar compounds with organic solvents. Examples of separations and chromatograms for its use in both aqueous and organic eluants are given.

Thin-layer chromatographic separation of intermediates of the pyridine nucleotide cycle

A rapid thin-layer chromatographic procedure for separation of the compounds comprising the intermediates in the salvage pathway known as the pyridine nucleotide cycle plus quinolinic acid and the reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate is described. The method utilizes silica gel high-performance thin-layer plates and a mobile phase of methanol, tetrabutylammonium hydroxide, and acetonitrile. The time required for analysis is greatly reduced and results in greater than 96% purity of each migrating compound.

Improvement of the chemical analysis of antibiotics I. Simple method for the analysis of tetracyclines on silica gel high-performance thin-layer plates

A technique for determination of tetracyclines using silica gel high-performance thin-layer chromatography followed by densitometry is established. A predevelopment with saturated disodium ethylenediaminetetraacetate aqueous solution and the complementary use of three solvent systems enable a reliable identification. Direct determination using densitometry without further treatment with any reagents is of high sensitivity and reproducibility. A change of measurement wavelength enables the determination of overlapping spots.

More economical use of high-performance thin-layer plates for chromatographic screening of illicit drug samples

A rapid thin-layer chromatographic method for the separation of illicit drugs and similar compounds by two simultaneous but different developments on the same chromatographic plate is described. One half of the plate was impregnated with an aqueous solution of KHSO 4 and the whole plate developed in a Camag linear developing chamber from two opposite sides using methanol with 0.01 M KBr as eluent.

Densitometric quantification of brain gangliosides separated by two-dimensional thin layer chromatography

A procedure for accurate densitometric quantification of gangliosides separated by two-dimensional thin layer chromatography is reported. The procedure was set up employing 9 different pure gangliosides and was applied to the analysis of calf and pig brain gangliosides. Silica gel high performance thin layer plates, 10 x 10 cm. were two-dimensionally developed at 18-20 C with the following solvents: chloroform methanol 0.2% aqueous CaCl(2), 50/40/10 by volume, for the first run; n-propanol 17 M NH(4)OH/water, 6/2/1 by volume for the second run. Ganglioside spots were visualized by spraying with an Ehrlich reagent, which is specific for sialic acid, and heating at 120 C for 15 min. The spots were quantified by sequential scanning densitometry, linear responses being obtained for ganglioside amounts on the plate ranging from 0.1 to 6 nmol as bound sialic acid. The reproducibility of densitometric responses resulted to be acceptable since the standard deviation values were lower than +/- 15% of the mean values also for those ganglioside species contained in minor proportions. The ganglioside mixtures of calf and pig brain were resolved in about 20 spots. Of these 9 corresponded to gangliosides GM3, GM2, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b, which were identified with certainty and quantified. The identification of GM3 (carrying N-glycolylneuraminic acid), GD3, GD1a (carrying N-acetyl- and N-glycolyl-neuraminic acid) and GT1a was only tentative. All the other spots corresponded to unidentified gangliosides, some of them possibly new species.