Thin-layer Chromatography Results Research Articles

Chromatographic fingerprinting and free-radical scavenging activity of ethanol extracts of Muntingia calabura L. leaves and stems

ObjectiveTo determine the thin-layer chromatography (TLC) fingerprint profiles and to evaluate the in vitro antioxidant activity of ethanol extracts of Muntingia calabura (M. calabura) leaves and stems. MethodsThe leaves and stems were extracted using ethanol as solvent. The TLC separation of the phytochemical constituents of the leaf and ethanol extracts was carried out in ethyl acetate: n-hexane and chloroform: ethyl acetate mobile phase systems. Distinct spots were visualized under visible light, UV 254 nm, UV 366 nm and after spraying with vanillin-sulfuric acid. The 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging assay was used to evaluate the antioxidant activity of the extracts. ResultsBoth the leaf and stem ethanol extracts at 4 mg/mL exhibited 2,2-diphenyl-1-picrylhydrazyl inhibition of more than 90%, relative to gallic acid. The results of TLC showed that the degree of resolution between the constituent spots was comparable between the two mobile phase systems using the different visualization wavelengths. Under the 254 nm visualization, few spots were observed in leaf and stem extracts. Visualization at 366 nm yielded the greatest number of observable spots of various colors in both leaf and stem extracts. More spots were visualized upon post-derivatization with vanillin-sulfuric acid in the TLC chromatograms using chloroform: ethyl acetate mobile phase, compared to those in ethyl acetate: n-hexane mobile phase. ConclusionsM. calabura exhibited very high antioxidant activity in its leaves and stems ethanol extracts, both of which are used in traditional medicine. The TLC results demonstrated the presence of diverse secondary metabolites in the leaf and stem ethanol extracts, indicating that the antioxidant activity, including other bioactivities may be attributed to these phytochemical constituents. This paper has reported for the first time the TLC fingerprinting of M. calabura using visible light, UV 254 nm, UV 366 and post-derivatization with vanillin-spray to visualize separate spots on TLC plates.

Isolation of endophytic fungi from Macleaya cordata and screening of sanguinarine-producing strains

Endophytic fungi were isolated from Macleaya cordata growing in Dabie Mountain by agar-block method, and then the endophytic fungi were grouped into different types based on their morphological characteristics, and thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were employed to determine whether the metabolic substances contained sanguinarine or not, and then preliminarily identified by morphological method. The results showed that the leaves hosted the largest number of endophytes (96 isolates) followed by the stems (57 isolates) and finally the roots (28 isolates), respectively. Based on morphological characteristics the endophytic fungi were grouped into 26 types in our study. TLC and HPLC results showed that there was sanguinarine in the metabolic substances of BLH 51 strain. According to the morphological characteristic, the BLH 51 strain was identified as Fusarium proliferatum. All these indicated that the medicinal plant M. cordata harbors abundant endophytes, which could be a new source for the search of active secondary metabolites.

Mucor fragilis as a novel source of the key pharmaceutical agents podophyllotoxin and kaempferol

Context: Podophyllotoxin, a pharmaceutically important bioactive compound of Podophyllum sps. (Berberidaceae), is in great demand worldwide as an anticancer and antivirus drug precursor. However, the source of podophyllotoxin is very limited due to the endangered status of the Podophyllum plant.Objective: The aim of this study was to isolate podophyllotoxin-producing endophytic fungi from Sinopodophyllum hexandrum (Royle) Ying (1979) (Berberidaceae) plants of the Taibai Mountains of China in order to obtain bioactive compounds.Materials and methods: The strains producing kaempferol and podophyllotoxin were screened by thin-layer chromatography (TLC) analysis. The presence of kaempferol and podophyllotoxin in extracts of these strains was further confirmed by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) analyses.Results: Among six endophytic fungi isolated from the rhizomes of S. hexandrum, one strain was able to produce kaempferol. Another strain, named TW5, was able to produce both kaempferol and podophyllotoxin simultaneously according to the TLC, HPLC, and NMR results. The podophyllotoxin yield of TW5 was calculated to be 49.3 μg/g of mycelial dry weight after 7-d fermentation. Strain TW5 was identified morphologically and phylogenetically to be Mucor fragilis Fresen. (Mucoraceae). These results suggest that the podophyllotoxin-synthesizing ability is obtained by uptaking genes involved in the podophyllotoxin synthesis from the host plant into endophytic fungal genomes.Conclusion: Our results showed, for the first time, that the endophytic fungus M. fragilis is able to produce simultaneously the same two bioactive metabolites, podophyllotoxin and kaempferol, as its host plant. Furthermore, the relatively high podophyllotoxin yield obtained may improve the industrial production of podophyllotoxin, which may help protect this endangered plant.

Validation of Thin Layer Chromatography with AccuTOF-DART™ Detection for Forensic Drug Analysis*

Thin layer chromatography (TLC) is a technique that is commonly employed in the forensic drug analysis of pharmaceutical preparations. Detection is typically accomplished using various visualization spray reagents. Conventional gas chromatography-mass spectrometry (GC-MS) analysis is typically performed to confirm the TLC results. Depending on the drugs tested and the instrument conditions required, this confirmation can take up to an hour to complete. Direct analysis in real time (DART™) is an ionization source, coupled to an accurate-mass time-of-flight mass spectrometer that has the capability to ionize materials under ambient conditions. To streamline analysis, the combination of TLC with DART™ detection is proposed to screen and subsequently identify drug compounds, all from the same TLC plate. DART™ confirmations of TLC analyses take <10 min to complete and compare favorably to GC-MS in sensitivity and selectivity. This study validates the use of TLC-DART in the forensic identification of the components of several pharmaceutical preparations.

Co-occurrence of Aflatoxins, Ochratoxin A, Fumonisins, and Zearalenone in Cereals and Feed, Determined by Competitive Direct Enzyme-Linked Immunosorbent Assay and Thin-Layer Chromatography

Aspergillus, Penicillium, and Fusarium species frequently contaminate crops. For this reason mycotoxins such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), and zearalenone (ZEA) are found in food and feed in a wide range of concentrations, depending on environmental and storage conditions. Consumption of mycotoxin-contaminated food and feed has been associated with acute and chronic poisoning and carcinoma. The aim of this study was to determine the incidence and co-occurrence of AFs (B1+B2+G1+G2), OTA, FBs (B1+B2+B3), and ZEA in 37 samples of cereals and feed randomly collected in 2007 from households of an endemic nephropathy (EN) area in Croatia. The mycotoxins were determined using the competitive direct ELISA test (CD-ELISA) in combination with thin-layer chromatography (TLC). The most frequent mycotoxin was ZEA (92%, mean 318.3 microg kg-1), followed by FBs (27%, 3690 microg kg-1), AFs (24.3%, 4.6 microg kg-1), and OTA (16.2%, 9.8 microg kg-1). Levels of AFs, ZEA, and FBs detected by CD-ELISA significantly correlated with the TLC results. However, only one OTA-positive sample was confirmed by TLC due to its high limit of detection. The levels of these mycotoxins were below the permissible limit for animal feed. Twenty-nine percent of cereals were contaminated with FBs, OTA, or ZEA in mass fractions above the permissible limit for humans. Co-occurrence of two toxins varied between 4.2% and 54% and of three between 4.2% and 7.6%. Prolonged co-exposure to AFs, OTA, FBs, and ZEA might increase the risk of various chronic diseases.

Therapeutic Drug Monitoring of Nevirapine in Resource‐Limited Settings

We developed a simple and inexpensive thin-layer chromatography (TLC) assay for semiquantitative detection of saliva concentrations of nevirapine in resource-limited settings. The method was validated in an African target population. Paired plasma and saliva nevirapine concentrations were assayed by high-performance liquid chromatography (HPLC); saliva concentrations of nevirapine were also assayed by TLC. The rate of false-positive results was the proportion of subtherapeutic nevirapine saliva and plasma concentrations determined by HPLC that were judged to be therapeutic in saliva specimens by TLC. The rate of false-negative results was the proportion of therapeutic nevirapine saliva and plasma concentrations determined by HPLC that were judged to be subtherapeutic in saliva specimens by TLC. The extent of agreement in TLC readings between 5 technicians and 2 batches of TLC sheets was evaluated. Twenty-five (9%) of 286 African adults had a subtherapeutic plasma nevirapine concentration. The median ratio of nevirapine concentrations in saliva to those in plasma was 0.51:1. The rate of false-positive results for TLC was 0% (0 of 23 specimens) when TLC results were compared with HPLC results for saliva specimens and 8% (2 of 25 specimens) when TLC results were compared with HPLC results for plasma specimens. The rate of false-negative results for TLC was 1% (3 of 263 specimens) when TLC results were compared with HPLC results for saliva specimens and 1% (3 of 261 specimens) when TLC results were compared with HPLC results for plasma specimens. The extent of agreement of TLC results was substantial for the 5 technicians (Fleiss's kappa = 0.77) and for the 2 batches of sheets (Cohen's kappa = 0.80). The TLC assay was found to be sensitive, specific, and robust in the detection of subtherapeutic nevirapine concentrations in saliva specimens obtained from African HIV-infected adults. It is an attractive alternative to HPLC for therapeutic drug monitoring of nevirapine in resource-limited settings.

Using Laboratory Chemicals To Imitate Illicit Drugs in a Forensic Chemistry Activity

This forensic chemistry activity utilizes presumptive forensic testing procedures and laboratory chemicals that produce screening results similar to controlled substances. For obvious reasons, obtaining heavily regulated controlled substances to create an undergraduate student activity is not practical for most educational institutions. We were able to identify over-the-counter and laboratory chemicals that mimic actual street drugs in terms of physical properties and color response. Using these selected chemicals, the screening aspect of drug testing provides students with the opportunity to understand what obstacles a forensic chemist faces when analyzing a sample of unknown identity. Chemical spot tests (CSTs) and thin-layer chromatography (TLC) were chosen owing to their simplicity, versatility, common use by forensic chemists, availability, and cost of the reagents and equipment needed. Students are expected to explain how simple color reactions can screen for illicit drugs, to describe how TLC can be used to separate and tentatively identify drugs, to predict how polarity changes affect TLC results, and to explain why CSTs and TLC serve for screening purposes only.

A possible cellular explanation for the NMR-visible mobile lipid (ML) changes in cultured C6 glioma cells with growth

The NMR-visible mobile lipid (ML) signals of C6 glioma cells have been monitored at 9.4 and 11.7 T (single pulse and 136 ms echo time) from cell pellets by 1H NMR spectroscopy. A reproducible behavior with growth has been found. ML signals increase from log phase (4 days of culture) to postconfluence (7 days of culture). This ML behavior is paralleled by the percentage of cells containing epifluorescence detectable Nile Red stained cytosolic droplets (range 23%–60% of cells). The number of positive cells increases after seeding (days 0–1), decreases at log phase (days 2–4), increases again at confluence (day 5) and even further at post-confluence (day 7). C6 cells proliferation arrest induced by growth factors deprivation induces an even higher accumulation of cytosolic droplets (up to 100% of cells) and a large ML increase (up to 21-fold with respect to 4-day log phase cells). When neutral lipid content is quantified by thin-layer chromatography (TLC) on total lipid extracts of C6 cells, no statistically significant change can be detected (in μg/10 8 cells) with growth or growth arrest in major neutral lipid containing species (triacylglycerol, TAG, diacylglycerol, DAG, cholesteryl esters, ChoEst) except for DAG, which decreased in post-confluent, 7-day cells. The apparent discrepancy between NMR, optical microscopy and TLC results can be reconciled if possible biophysical changes in the neutral lipid pool with growth are taken into account. A cellular explanation for the observed results is proposed: the TAG-droplet-size-change hypothesis.

FS01.3 Disperse (yes), orange (yes), 3 (no): what do we test in textile dye dermatitis?

Introduction: Patients sensitized to para‐phenylenediamine (PPD) have a high degree of patch test reactivity to Disperse Orange 3 (DO3), and a lesser one to Disperse Red 1 and Red 17. Two successive patients positive to PPD, Disperse Red 1 and 17, negative to DO3 were real eye‐openers for our considerations about purity of our current allergen DO3.Materials and methods: We realized comparative thin‐layer chromatography (TLC), with DO3 from Chemotechnique®(DO3‐Chem) and Trolab®(both extracted from petrolatum), and “pure” DO3 from two chemical providers. TLC clearly indicated that DO3‐Chem was not DO3. HPLC analysis with pure DO3 from Chemotechnique® and comparison of structures by NMR with samples of DO3, revealed that DO3‐Chem was Disperse Orange 31 (DO31). In addition, signals through the GERDA network allowed the collection of test materials and observations. Among other members, only 2 used DO3‐Chem (from 2 different batches) that was DO31 too, according to TLC Results: According to their data, they observed no or a lower reactivity to DO3 than expected (4 patients DO3‐Chem + among 23 PPD+ e.g.). Finally, the error was proved to be due to the provider of the dye to Chemotechnique®, who likely deleted the 1 of Disperse Orange 31 on his packaging.Discussion: Chemical structure of DO31 indicates a possible in vivo hydrolysis into nitroaniline and a second compound, a substituted PPD derivative that clearly does not frequently react in PPD positive patients. Like drugs, patch tests are submitted to post‐commercialization controls. In addition to allergens providers who should enhance their quality controls, dermato‐allergologists have to be vigilant, and must active networks when they observe a rare bird.

SEMIQUANTITATIVE ANALYSIS BY THIN‐LAYER CHROMATOGRAPHY (TLC) OF BIOGENIC AMINES IN DRIED, SALTED AND CANNED FISH PRODUCTS

AbstractA thin‐layer chromatography (TLC) method was tested using dried, salted and canned fish products for the identification and semiquantification of biogenic amines: histamine, cadaverine, putrescine, tryptamine, and tyramine. The solvent system was acetone: NH4OH (95:5) on a precoated silica gel plate, and the developing reagent was 0.2% ninhydrin with 2% acetic acid in methanol. The TLC results were compared with liquid chromatography (LC). For LC levels between 6–9 mg%, TLC gave close to 5 mg%, and for samples with biogenic amines of 10–60 mg%, LC and TLC results are more similar to each other. In case of upper values (&gt; 60 mg%) other methods have to be used or sample dilution is suggested. The proposed TLC was suitable to estimate: cadaverine (5‐40 mg%), and tyramine (10‐20 mg%) in dried salted, and cadaverine (10‐20 mg%), tryptamine (5‐10 mg%), and histamine (10‐60 mg%) in canned fish. With this TLC methodology is possible to screen samples with these amines levels, for routine quality control in laboratories.

A convenient method for the determination of the quality of goldenseal

Goldenseal ( Hydrastis canadensis) has emerged as one of the top five herbal supplements in the world-wide market. A convenient method for the determination of the quality and possible adulteration of goldenseal products is thin-layer chromatography (TLC). TLC analysis of 10 goldenseal samples using two solvent systems and spray reagents was conducted. Five of these samples contained both hydrastine and berberine, four contained berberine and one did not contain either of these alkaloids. These TLC results were verified by HPLC analysis.

Utility of thin layer chromatography for detection of opioids and benzodiazepines in a clinical setting

This study examined the utility of thin layer chromatography (TLC) for detection of recent use of opioids and benzodiazepines among drug addicts seeking treatment at the Drug Dependence Treatment Centre of All India Institute of Medical Sciences, New Delhi, India. Over a period of 5 years (1991–1995), 6,055 urine samples were analyzed for opioids (morphine, codeine, buprenorphine, dextropropoxyphene, pentazocine) and benzodiazepines (diazepam, nitrazepam) by TLC. Out of all the drug tests ( n = 9,922) carried out, 24% of the drugs had been used during the past 72 hr. Averaged across all drugs, the detection rates corresponding to 24, 48, and 72 hr by TLC were 37%, 36%, and 31%, respectively. A high percentage of negative TLC results was observed in these samples. Moderate sensitivity of the TLC assay procedure, low consumption of drug, short time between drug use and urine collection, overreporting by the subjects, and drug use history of the subject obtained from multiple sources led to high negative results. These findings suggest that all the TLC negative results also need further confirmation by an alternative, more sensitive technique in a clinical setting. This will make the drug abuse testing program more meaningful.

99/00233 Thin layer chromatography study of heavy liquefaction products derived from two Chinese bituminous coals

Two Chinese bituminous coals, Yanzhou and Fenxi, were liquefied in the temperature range of 375--450 C and under a cold H{sub 2} pressure of 7 MPa without the presence of a solvent. An iron sulfide catalyst, prepared by in-situ precipitation, was used in the study. Heavy liquefaction products, a portion of toluene and trichloroethylene (TCE) solubles, were studied by thin layer chromatography (TLC) technique. Under most conditions, the conversions of catalytic liquefaction are about twice as much as that of thermal liquefaction. The yields to toluene solubles are similar to that of TCE solubles. The TLC results of the heavy liquefaction products are compared with petroleum derived highway asphalts and with a coal tar pitch. The results show that the liquefaction products of Yanzhou coal, under certain conditions, have similar composition as that of petroleum derived highway asphalts, but significantly different from that of coal tar pitch, paraffinic petroleum residue and building asphalt.

Assay and purity control of minocycline by thin-layer chromatography using UV and fluorescence densitometry — a comparison with liquid chromatography

A thin-layer chromatography (TLC) method using UV and fluoresecence densitometry is described for the assay and purity control of minocycline (MC). With a mobile phase dichloromethane-methanol-water (57:35:8, v/v/v) and a silica gel thin-layer, previously sprayed with 10% m/v sodium edetate adjusted to pH 9.0, 4-epiminocycline and 7-didemethylminocycline were well separated from MC and from each other, 7-monodemethylminocycline and 6-deoxy-6-demethyltetracycline (6-DODMTC) were not separated from each other and were only partially separated from minocycline. 6-DODMTC was selectively determined by fluorescence densitometry, while quantification of other impurities and the assay of MC were performed by UV densitometry. Results obtained with qualitative TLC were compared with those obtained by a liquid chromatography (LC) method using a poly(styrene-divinylbenzene) copolymer stationary phase. The correlation coefficient for TLC and LC results was >0.999. For TLC the relative standard deviation for the assay of MC at 1.25 mg ml −1 was < 3.0% ( n = 4), while for LC it was < 1.0% ( n = 4).

Comparison of Two Immunochemical Methods with Thin-Layer Chromatographic Methods for Determination of Aflatoxins

Three different methods were compared for the determination of total flatoxins in corn and peanuts naturally contaminated with aflatoxins and in corn, peanuts, cottonseed, peanut butter, and poultry feed spiked with aflatoxins B1, B2, and G1. The 3 methods were an enzyme-linked immunosorbent assay (ELISA) screening test; a monoclonal antibody-affinity column-solid-phase separation method; and the AOAC official thin-layer chromatography (TLC) methods for all except poultry feed, for which Shannon's TLC method for mixed feed was used. The ELISA test is designed to provide only positive results for total aflatoxins at greater than or equal to 20 ng/g or negative results at less than 20 ng/g. The affinity column separation is coupled with either bromination solution fluorometry to estimate total aflatoxins or liquid chromatography (LC) to quantitate individual aflatoxins. Fluorodensitometry was used to determine aflatoxins in commodities analyzed by the TLC methods. The LC and TLC results were in good agreement for all the analyses. The results for the affinity column using bromination solution fluorometry were similar except those for cottonseed, which were about 60% higher. The ELISA screening method correctly identified naturally contaminated corn and peanut positive samples. No false positives were found for controls. The correct response for spiked corn, raw peanuts, peanut butter, and cottonseed at greater than or equal to 20 ng aflatoxins/g was about 90%. The correct response for spiked poultry feed at greater than or equal to 20 ng aflatoxins/g was about 50%.

Laboratory and clinical evaluation of a rapid slide agglutination test for phosphatidylglycerol

Amniostat-FLM is a recently developed rapid slide agglutination test for amniotic fluid phosphatidylglycerol. We compared phosphatidylglycerol quantitated by two-dimensional thin-layer chromatography with phosphatidylglycerol determined by Amniostat-FLM in 180 amniotic fluid samples. If any level of phosphatidylglycerol by thin-layer chromatography, including a trace, was considered positive, then phosphatidylglycerol by thin-layer chromatography and Amniostat-FLM were concordant in 171 of 180 samples (95%). Both were positive in 123 and both negative in 48 samples. The nine amniotic fluid samples with discrepant results were as follows: Five negative Amniostat-FLM results were associated with trace phosphatidylglycerol by thin-layer chromatography (1% to 2% of total phospholipids), one negative Amniostat-FLM result was associated with 3% phosphatidylglycerol by thin-layer chromatography, and three positive Amniostat-FLM results were associated with negative phosphatidylglycerol by thin-layer chromatography results. There were 14 samples with trace phosphatidylglycerol by thin-layer chromatography; of these, there were nine positive and five negative Amniostat-FLM results. If >2% phosphatidylglycerol by thin-layer chromatography was considered positive, concordance between the two tests was 93% (167 of 180). From these samples, 119 infants were delivered within 72 hours of amniocentesis. There were 100 positive Amniostat-FLM results and all were associated with the absence of respiratory distress syndrome; of the 19 negative Amniostat-FLM results, five were associated with infants who developed respiratory distress syndrome. The present study confirms that Amniostat-FLM is a sample, rapid, and accurate test for determining the presence of phosphatidylglycerol in amniotic fluid.

Evaluation of the rapid hippurate hydrolysis test with enterococcal group D streptococci

The rapid hippurate hydrolysis test was evaluated with the conventional test, using 17 group A streptococci, 9 non-enterococcal group D streptococci, 108 enterococcal group D streptococci, and 2 strains of Listeria monocytogenes. There was complete correlation between the rapid and conventional tests with all organisms except enterococcal group D. The rapid hippurate hydrolysis method was more sensitive with the enterococci; 95.4% were positive with the rapid method, and 9.3% were positive with the conventional method. Thin-layer chromatography (TLC) was performed on all isolates to determine if the end product of hydrolysis, glycine, was indeed present. The TLC results were in agreement with the rapid and conventional methods for group A streptococci, nonenterococcal group D streptococci, and L. monocytogenes. TLC results were in total agreement with the rapid hippurate hydrolysis test for the enterococcal group D isolates, thus verifying the accuracy of this more sensitive test. Trace amounts of glycine were found in the substrate, indicating the need for including an uninoculated substrate control as well as stock strains of group A and B beta-hemolytic streptococci (negative and positive controls, respectively) each time the rapid hippurate hydrolysis test is performed.