Thin-layer Chromatograms Research Articles

Structural Profiling of Individual Glycosphingolipids in a Single Thin-Layer Chromatogram by Multiple Sequential Immunodetection Matched with Direct IR-MALDI-o-TOF Mass Spectrometry

The thin-layer chromatography (TLC) immunoenzyme overlay assay is a widely used tool for antibody-mediated identification of glycosphingolipids (GSLs) in mixtures. However, because the majority of GSLs is left unexamined in a chromatogram of a single assay, we developed a novel method that permits detection of various GSLs by sequential multiple immunostaining combined with individual coloring of GSLs in the same chromatogram. Specific staining was achieved by means of primary anti-GSL antibodies, directed against lactosylceramide, globotriaosylceramide, and globotetraosylceramide, in conjunction with alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibodies together with the appropriate chromogenic substrates. Triple coloring with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-AP, Fast Red-AP, and 3,3'-diaminobenzidine (DAB)-HRP resulted in blue, red, and black precipitates, respectively, following three sequential immunostaining rounds. Structures of antibody-detected GSLs were determined by direct coupling of TLC with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. This combinatorial technique was used to demonstrate structural GSL profiling of crude lipid extracts from human hepatocellular cancer. This powerful technology allows efficient structural characterization of GSLs in small tissue samples and marks a further step forward in the emerging field of glycosphingolipidomics.

An epidemic of furniture-related dermatitis: searching for a cause

Background Sitting in new chairs or sofas has elicited dermatitis in numerous patients in Finland and in the U.K. since autumn 2006. The cause of the dermatitis seemed to be an allergen in the furniture materials. Objectives To determine the cause of the dermatitis in patients with furniture-related dermatitis. Methods Altogether 42 patients with furniture-related dermatitis were studied. First, 14 Finnish patients were patch tested with the standardized series and with the chair textile material. A thin-layer chromatogram (TLC) strip and an extract made from the same textile material were tested in seven Finnish patients. The test positive spot of the TLC and the content of a sachet found inside a sofa in the U.K. were analysed by using gas chromatography-mass spectrometry. All chemicals analysed were patch tested in 37 patients. Results A positive patch test reaction to the chair textile and to its extract was seen in all patients tested, one-third of whom had concurrent reactions to acrylates. Positive reactions to the same spot of the TLC strip were seen in five of seven patients and dimethyl fumarate was analysed from the spot as well as from the sachet contents. Dimethyl fumarate (0.01%) elicited positive reactions in all the patients. The other chemicals analysed did not elicit positive reactions, but one patient in the U.K. had a positive reaction to tributyl phosphate. Conclusions Sensitization to dimethyl fumarate was seen in all the patients with furniture-related dermatitis. Concurrent sensitization or cross-reactions were common among the sensitized patients.

Patch testing of patients allergic to Disperse Blue 106 and Disperse Blue 124 with thin‐layer chromatograms and purified dyes

Former investigations have demonstrated that patch test preparations containing Disperse Blue (DB) 106 and DB 124 are impure. To investigate the significance of impurities in preparations of disperse dyes with regard to contact allergy. 21 patients allergic to DB 106 and/or DB 124 were tested with dilution series of commercial and purified DB 106 and DB 124. All patients were additionally tested with thin-layer chromatography (TLC) strips made from the commercial preparations containing DB 106 and 18 of them also with TLC strips containing DB 124. 13 of 21 and 12/18 patients tested positively to the strips of DB 106 and DB 124, respectively. Among these patients, 4/13 and 5/12, respectively, did not react to the main spot but reacted to other spots. Dilution series of purified DB 106 and DB 124 were positive in 10 patients each, while 16 and 15 patients, respectively, tested positively to the dilution series of the corresponding commercial disperse dye. Approximately 25% of the patients, diagnosed as contact allergic to DB 106 and DB 124, only reacted to impurities in the patch test preparations. This has implications for diagnosis and prevention.

Norwalk virus-like particles bind specifically to A, H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids

Noroviruses and norovirus virus-like particles (VLPs) exhibit strain specific patterns in their binding to ABH and Lewis histo-blood group antigens. In this study we demonstrate for the first time specific binding of Norwalk virus VLPs to type 1 and type 2 chain glycosphingolipids (GSLs) carrying ABH and Lewis antigens. N-succinimidyl-3-tributylstannyl benzoate (ATE) was precursor labeled with (125)I and then conjugated to VLPs. The (125)I-VLPs were used in GSL thin-layer chromatogram binding assays and displayed binding to H type 1, Lewis b, A type 1, A Lewis b GSLs but no binding to B type 1 or B Lewis b GSLs. For the type 2 chain GSLs the Norwalk VLPs bound to H type 2, Lewis y, A type 2 and A Lewis y. In addition, the VLPs bound to several complex GSLs from blood group O and A, but not from blood group B red blood cells.

Antibacterial Activities of Dodonaea viscosa using Contact Bioautography Technique

The crude ethanolic extract and n-hexane, dichloromethane, ethyl acetate, n-butanol and aqueous fractions of Dodonaea viscosa were analyzed for antibacterial potential against four Gram positive bacteria: Bacillus subtilis, Bacillus cereus, Micrococcus luteus, Staphylococcus aureus, and three Gram negative bacteria: Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa. Preliminary screening showed inhibition against Staphylococcus aureus, Micrococcus luteus, Escherichia coli and Pseudomonas aeruginosa. The thin layer chromatograms of the fractions were then subjected to contact bioautography, which showed inhibition zone at different Rf values against Bacillus subtilis, Micrococcus luteus, Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa, indicating the presence of antibacterial components. The MIC of each fraction was determined through a 96-well micro-titer plate method. The non-viability of the organisms was ascertained by determining the MBC of the fractions.

Sulfatide Recognition by Colonization Factor Antigen CS6 from Enterotoxigenic Escherichia coli

The first step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections is adhesion of the bacterium to the small intestinal epithelium. Adhesion of ETEC is mediated by a number of antigenically distinct colonization factors, and among these, one of the most commonly detected is the non-fimbrial adhesin coli surface antigen 6 (CS6). The potential carbohydrate recognition by CS6 was investigated by binding of recombinant CS6-expressing E. coli and purified CS6 protein to a large number of variant glycosphingolipids separated on thin-layer chromatograms. Thereby, a highly specific binding of the CS6-expressing E. coli, and the purified CS6 protein, to sulfatide (SO3-3Galβ1Cer) was obtained. The binding of the CS6 protein and CS6-expressing bacteria to sulfatide was inhibited by dextran sulfate, but not by dextran, heparin, galactose 4-sulfate or galactose 6-sulfate. When using recombinantly expressed and purified CssA and CssB subunits of the CS6 complex, sulfatide binding was obtained with the CssB subunit, demonstrating that the glycosphingolipid binding capacity of CS6 resides within this subunit. CS6-binding sulfatide was present in the small intestine of species susceptible to CS6-mediated infection, e.g. humans and rabbits, but lacking in species not affected by CS6 ETEC, e.g. mice. The ability of CS6-expressing ETEC to adhere to sulfatide in target small intestinal epithelium may thus contribute to virulence.

Comparison of wheat varieties on the basis of their relative phenolic contents

Fluorescence and absorption curves obtained from one dimension thin-layer phenol chromatograms of eight varieties of wheat were used in an attempt to characterize the varieties. By comparing the ratios of some specific peaks (A/D and A + C + D/B2) between the varieties and in both kinds of curves, consistent differences between some of the varieties could be obtained. By plotting the varietal values obtained from the two kinds of peak ratios in a two-way diagram it was possible to separate out the varieties Chinese Spring and Maris Ensign. The four varieties Peko, Capa, Norko and Aster were found to form a distinct group with all the varieties in the group having low A/D and A + C + D/B2 ratios. The varieties Pondus and Østby formed a second group having high A/D and high A + C + D/B2 ratios. There was good agreement between the fluorescence and absorption measurements and both the methods served to confirm and complement one another.

Spectrophotometric and spectrodensitometric determination of Clopidogrel Bisulfate with kinetic study of its alkaline degradation

Four sensitive, selective and precise stability-indicating methods for the determination of Clopidogrel Bisulfate (CLP) in presence of its alkaline degradate and in pharmaceutical formulations were developed and validated. Method A is a second derivative (D 2) spectrophotometric one, which allows the determination of CLP in presence of its alkaline degradate at 219.6, 270.6, 274.2 and 278.4 nm (corresponding to zero-crossing of the degradate) over a concentration range of 4–37 μg mL −1 with mean percentage recoveries 99.81 ± 0.893, 99.72 ± 0.668, 99.88 ± 0.526 and 100.46 ± 0.646, respectively. CLP can be determined in the presence of up to 65% of its degradate. D 2 method was used to study the kinetic of CLP alkaline degradation that was found to follow a first-order reaction. The t 1/2 was 6.42 h while K (reaction rate constant) was 0.1080 mol/h. Method B is the first derivative of the ratio spectra (DD 1) spectrophotometric method, by measuring the peak amplitude at 217.6 and 229.4 nm using acetonitrile and CLP can be determined in the presence of up to 70% of its degradate. The linearity range was 5–38 μg mL −1 with mean percentage recoveries 99.88 ± 0.909 and 99.70 ± 0.952, respectively. Method C based on the determination of CLP by the bivariate calibration depending on simple mathematic algorithm which provides simplicity and rapidity. The method depends on quantitative evaluation of the absorbance at 210 and 225 nm over a concentration range 5–38 μg mL −1 with mean percentage recovery 99.27 ± 1.115. CLP can be determined in the presence of up to 70% of its degradate. Method D is a TLC-densitometric one, where CLP was separated from its degradate on silica gel plates using hexane:methanol:ethyl acetate (8.7:1:0.3, v/v/v) as a developing system. This method depends on quantitative densitometric evaluation of thin layer chromatogram of CLP at 248 nm over a concentration range of 0.6–3 μg/band with mean percentage recovery 99.97 ± 1.161. CLP can be determined in the presence of up to 90% of its alkaline degradate. The selectivity of the proposed methods was checked using laboratory prepared mixtures. The proposed methods have been successfully applied to the analysis of CLP in pharmaceutical dosage forms without interference from other dosage form additives and the results were statistically compared with the official method.

Forensic discrimination of blue ballpoint pen inks based on thin layer chromatography and image analysis

This article aims to provide a new and fast method for differentiation of inks on a questioned document. The data acquisition was carried out by designing specific image analysis software for evaluating thin layer chromatograms (TLC-IA). The ink spot was extracted from the document using methanol and separated by TLC using plastic sheet silica gel 60 without fluorescent indicator, and a mixture of ethyl acetate, ethanol, and water (70:35:30, v/v/v) as mobile phase. To discriminate between different pen inks, new software was designed on the basis of intensity profile of red, green, and blue (RGB) characteristic. In practice, after development of chromatogram, the chromatograms were scanned by ordinary office scanner, intensity profiles of RGB characteristics on the development straight of each sample were produced and compared with the mentioned software. RGB profiles of ballpoint inks from various manufacturers showed that the patterns in most cases were distinctly different from each other. This new method allowed discriminating among different pen inks with a high reliability and the discriminating power of 92.8%. Blue ballpoint pen inks of 41 different samples available on the local market were successfully analyzed and discriminated.

Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and GDlb) in a patient with motor neuron syndrome

Small amounts of oligoclonal immunoglobulins were detected by Western blotting in the serum from a patient with motor neuron syndrome. The prominent one, a monoclonal IgM lambda, reacted strongly with the gangliosides GM1 and GD1b and more weakly with asialo GM1, as shown by immunoenzymatic staining of thin-layer chromatograms of gangliosides, ELISA on purified glycolipid coats and immunoadsorption with purified GM1. Affinity-chromatography with purified GM1 resulted in the purification of monoclonal IgM lambda. This purified IgM and its Fab fragments showed the same pattern of reactivity with gangliosides as that observed with whole serum. Such monoclonal IgM could be responsible for motor neuron diseases in some patients with overt or barely detectable monoclonal gammopathies.

Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells

Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.

Glycolipid Studies in Small Intestine and Pancreas of α1,3-Galactosyltransferase Knockout Miniature Swine: α1,3GALT-KO Animals Lack αGAL Antigens and Contain Novel Blood Group H Compounds

Background To avoid hyperacute rejection of xeno-organs, α1,3-galactosyltransferase knock-out (GalT-KO) pigs have been produced. However, Galα1,3Gal (Gal) determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens that might interfere with the human immune response. Methods Glycolipids isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig were tested for immune reactivity with antibodies on thin-layer chromatograms after separation by high-performance liquid chromatography, and selected fractions were analysed by proton NMR spectroscopy. Results Immunostaining using purified human anti-Gal Abs revealed that tissues from WT animals express large amounts of Gal-antigens whereas GalT-KO tissues lacked these antigens. Proton NMR spectroscopy on small intestine fractions revealed both linear and branched nona- and decaglycosylceramides, respectively, with terminal Gal-epitopes. In corresponding GalT-KO fractions, Gal-epitopes seemed to be replaced by terminal α1,2fucoses. Two novel branched blood group H compounds was found in the GalT-KO intestine. Conclusions The structural complexity of αGal-terminating antigens in the WT organs is very high. Knockout of α1,3GalT by gene-targeting results in elimination of Gal-determinants. In addition structurally novel α1,2fucose-terminated blood group H compounds were identified in the GalT-KO tissue. These compounds are not expected to be recognized by the human immune system.

Glycolipid binding epitopes involved in adherence of the periodontitis-associated bacterium Porphyromonas gingivalis

The ability of the periodontal pathogen Porphyromonas gingivalis to use different glycolipid structures as receptors has previously been demonstrated. The bacterium adhered to acid and nonacid glycolipids originating from human organs and to nonacid glycolipids of porcine origin. The aim of the present study was to analyze these binding epitopes by structural characterization. Glycolipid fractions with positive bacterial binding from e.g. human and porcine origin, were purified by the high performance liquid chromatography technique and thereafter used in bacterial overlay assays with (35)S-labeled P. gingivalis. Purified fractions with positive binding were structurally characterized by proton nuclear magnetic resonance spectroscopy. Complementing thin-layer chromatograms and bacterial overlay assays with pure reference glycolipid fractions and competition experiments with lactose were performed to define potential receptors. The P. gingivalis binding epitopes, including cerebrosides with nonhydroxy fatty acids, lactosylceramide with hydroxy fatty acids, sulfatides, lacto-, neolacto- and gangliotetraosylceramides, are in several instances similar to those found for other bacteria, e.g. H. pylori, H. influenzae and N. meningitidis. In addition P. gingivalis also bound to the Galalpha4Gal epitope of the globo series of glycolipids. In the future these results may be valuable for development of new treatment strategies, such as anti-adhesion therapies and vaccines specifically directed against P. gingivalis infection.

Mutation of Streptomyces griseoflavus in Order to Obtain High Yield Desferrioxamine Producing Fused Cells

Streptomyces griseoflavus PTCC 1130 was mutated by UV irradiation. Two mutants were obtained (C7031 and S7011). These two mutants were able to produce desferioxamine. Desferrioxamine was extracted from the culture broth of the two mutated strains and the thin layer chromatogram of the products showed the R(F) values of 0.461, 0.463 and 0.456 for S7011, C7031 and the standard, respectively. The protoplasts of mutated Streptomyces griseoflavus were isolated and fused together. Total numbers of 58 fusions were obtained and only 17 fusions showed significant resistance to sodium azide and crystal violet. In terms of production of desferrioxamine only fusion PF9 and PF10 increased 68.3 and 81.8% desferrioxamine production as compared to parent strain (PTCC 1130), respectively.

Studies on Glycolipid Antigens in Small Intestine and Pancreas from α1,3-Galactosyltransferase Knockout Miniature Swine

To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knockout (GalT-KO) pigs have been produced. Galalpha1,3Gal determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens. This is the first biochemical study of carbohydrate antigens in GalT-KO pig organs. Neutral and acidic glycolipids were isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig. Glycolipid immune reactivity was tested on thin-layer chromatograms. Small intestine neutral glycolipids were separated by high-performance liquid chromatography and selected fractions were analyzed by proton nuclear magnetic resonance spectroscopy. Total gangliosides were quantified on thin-layer chromatograms and in microtiter wells. Using Galalpha1,3nLc4 glycolipid reference, total Galalpha1,3Gal glycolipid antigens in the WT animal was estimated at about 30 microg (small intestine) and 3 microg (pancreas) per gram of dry tissue. Galalpha1,3Gal determinants were not detected in GalT-KO tissues at a detection limit of less than 0.25% (small intestine) and 0.5% (pancreas) of the WT tissues. Isoglobotriaosylceramide (iGb3) was absent but trace amounts of Fuc-iGb3 was found in both GalT-KO and WT pig small intestine. Blood group H type 2 core saccharide compounds were increased in GalT-KO pancreas. Total amount of gangliosides was decreased in GalT-KO tissues. The alpha1,3-galactosyltransferase acceptor, N-acetyllactosamine determinant, was not increased in GalT-KO tissues. Human serum antibodies reacted with WT organ Galalpha1,3Gal antigens and gangliosides, of which the ganglioside reactivity remained in GalT-KO tissues. Knockout of porcine alpha1,3-galactosyltransferase gene results in elimination of Galalpha1,3Gal-terminated glycolipid compounds. GalT-KO genetic modification did not produce new compensatory glycolipid compounds reactive with human serum antibodies.

Benzil and Benzoin: General Spray Reagents for the Visualization of Organic Compounds on Thin Layer Chromatograms

Benzil and Benzoin spray reagents have been adopted to detect some organic compounds on thin layer chromatograms (TLC). These reagents were found to visualize sugars, amino acids, dicarboxylic acids and some natural products on thin layer plates. Colors produced with these reagents were shades of the spectrum and characteristic of individual or class of the organic and natural products.

Patch testing with thin-layer chromatograms of chamomile tea in patients allergic to sesquiterpene lactones

Patients with contact allergy to sesquiterpene lactones (SLs) are usually hypersensitive to Asteraceae plant products such as herbal teas. The objective of this study was to show sensitizers in chamomile tea by patch testing with thin-layer chromatograms. Tea made from German chamomile was separated by thin-layer chromatography. Strips of the thin-layer chromatograms were used for patch testing SL-positive patients. 15 (43%) of 35 patients tested positively to 1 or more spots on the thin-layer chromatogram, with many individual reaction patterns. Patch testing with thin-layer chromatograms of German chamomile tea showed the presence of several allergens.

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus

Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-2), Manp(alpha1-->2)Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-3a), Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)-Ins-P-Cer (Af-3b), Manp(alpha1-->2)-Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)Ins-P-Cer (Af-4), and Manp(alpha1-->3)Manp(alpha1-->6)GlcpN(alpha1-->2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcNalpha1-->2Ins linkage may proceed by a two-step process, similar to the GlcNalpha1-->6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(beta1-->6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(beta1-->6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.

A dual biosensor for 2‐alkyl‐4‐quinolone quorum‐sensing signal molecules

Pseudomonas, Burkholderia and Alteromonas species produce diverse 2-alkyl-4-quinolones (AHQs) which inhibit the growth of bacteria, algae and phytoplankton, chelate iron, modulate mammalian host immune defences and act as quorum-sensing (QS) signal molecules. To facilitate the detection, identification and quantification of the major Pseudomonas aeruginosa AHQs 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) we developed two different AHQ biosensors. These were constructed by introducing either a lecA::luxCDABE or a pqsA::luxCDABE reporter gene fusion into a P. aeruginosa pqsA mutant which cannot synthesize AHQs. While both biosensors responded similarly to PQS (EC(50) 18 +/- 4 microM), the pqsA::luxCDABE biosensor was most sensitively activated by HHQ (EC(50) 0.44 +/- 0.1 microM). This biosensor was also activated albeit less sensitively by (i) PQS analogues with alkyl chains varying from C1 to C11, (ii) HHQ analogues with C9 and C11 alkyl chains and (iii) 2-heptyl-4-hydroxyquinoline-N-oxide (HHQNO). The AHQ biosensor also responded differentially to the AHQs present in cell free culture supernatants prepared from PAO1 and isogenic strains carrying mutations in genes (pqsA, pqsH, lasR, lasI, rhlR, rhlI) known to influence AHQ production. The AHQ profiles of P. aeruginosa strains was also evaluated by overlaying thin layer chromatogram (TLC) plates with the pqsA::luxCDABE biosensor. In PAO1, three major bioluminescent spots were observed which correspond to PQS, HHQ and a mixture of 2 nonyl-4-quinolone and HHQNO. We also noted that on TLC plates the biosensor not only produced bioluminescence in response to AHQs but also the green pigment, pyocyanin which offers an alternative visual indicator for AHQ production.

Risks and possibilities in patch testing with contaminated personal objects: usefulness of thin‐layer chromatograms in a patient with acrylate contact allergy from a chemical burn

We report a case of a chemical burn from dipropylene glycol diacrylate (DPGDA) spilt on working shoes, followed by active sensitization, thus giving an occupational allergic contact dermatitis on the patient's dorsal feet. Diagnostic tests included patch testing with acetone extracts made from the different shoe layers and thin-layer chromatograms. An invisible spot on the thin-layer chromatography plate gave a test eczema and was further investigated with gas chromatography-mass spectrometry. DPGDA was detected in the spot.