One-dimensional Thin-layer Chromatography Research Articles

EFFECT OF N-STEAROYLETHANOLAMINE ON THE LIPID COMPOSITION OF THE FRONTAL CORTEX AND HIPPOCAMPUS OF THE RAT'S BRAIN AT THE AGING

Aim. To study the possible protective effect of cannabimimetic lipid - N-stearoylethanolamine (NSE) on the lipid composition of the frontal cortex, hippocampus and on the state of episodic memory of old rats. Methods. Extraction of lipids from the tissues of the hippocampus and frontal cortex of rats was performed by the method of Bligh and Dyer. Phospholipids were separated by two-dimensional thin layer chromatography. Methyl esters of fatty acids from lipid extract were obtained by a modified method of Carreau and Dubaco. Quantitative analysis of fatty acid methyl esters was performed by gas-liquid chromatography on an Agilent GC7890 chromatograph with an Agilent 8987 mass detector. The fractions of free and esterified cholesterol were separated by one-dimensional thin layer chromatography. The dry cholesterol residue was analyzed on a Carlo Erba gas-liquid chromatograph. Results. The study of the diacyl (DF) and plasmalogen (PF) forms of phospholipids (PLs) content in the frontal cortex and hippocampus have shown a significant decrease in the plasmalogen form of PE (Phosphatidylethanolamine) (up to 15%) and an increase in its DF, compare to its content in young rats. Administration of NSE to old rats led to a significant increase in PF PE and did not cause significant changes in the content of PF in the composition of other PL of the frontal cortex of the brain and hippocampus. The decrease in the percentage of various phospholipids was found in frontal cortex and hippocampus of old rats: the content of phosphatidylcholine (PC) and phosphatidylinositol (PI) was significantly reduced in the frontal cortex and the decrease of diphosphatidylglycerol (DPG), PI and phosphatidylserine (PS) was found in the hippocampus, compare to the young animals. Administration of NSE to old rats had a different effects on the content of various phospholipids. The increase in the content of PC and PI in the frontal cortex and PS and DPG in the hippocampus is particularly pronounced due to NSE. An increase in the content of saturated fatty acids (FFAs ) and a decrease in the content of unsaturated FFAs in the frontal cortex and hippocampus of old rats also has been found. It has also been found that NSE administration to old rats promoted the growth of the free cholesterol level in the frontal cortex and hippocampus. The results of the New Object Recognition test in old rats have shown that a short-term memory has been improved by NSE. Conclusions. The administration of NSE to old rats causes an increase in PF of PLs in the frontal cortex and hippocampus of the brain, which can be considered as one of the mechanisms of neuroprotective action of NSE in aging. The changes in the phospholipids and fatty acids composition, and free cholesterol level of the frontal cortex and hippocampus of the brain of old rats caused by NSE administration have been shown to be adaptive and restorative. The New Object Recognition Behavioral Test has shown that NSE restores short-term memory in older rats. The obtained results expand the understanding of the mechanisms of biological action of NSE during aging in mammals and create the basis for the development a new drug with geroprotective properties.

Detection of edible insect derived phospholipids with polyunsaturated fatty acids by thin-layer chromatography, gas chromatography, and enzymatic methods

Dietary phospholipid (PL) intake from various animals and plants accounts for about 10 % of total lipids; however, the presence of PLs and their species in edible insects have yet to be fully reported. We therefore investigated the quantity and quality of PLs in edible insects (crickets, migratory locusts, and silkworms) using thin-layer chromatography (TLC), gas chromatography (GC), and enzymatic methods. Separation of PLs from total lipids extracted by a Folch method (chloroform and methanol mixture) was achieved using a two-dimensional TLC method. Based on retention factor values, this method successfully separated phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine, lysophosphatidylcholine, and cardiolipin (CL). Crickets contained higher levels of choline-binding PLs than either migratory locusts or silkworms. Fatty acid profiles of triglyceride (TG) and PL fractions from a one-dimensional TLC method reflected those of total lipids from GC analysis. Crickets were rich in TGs and PLs (PC and PE/CL), which bound n-6 polyunsaturated fatty acids (PUFAs), whereas migratory locusts and silkworms were rich in TGs and PE/CL, which bound n-3 PUFAs. A modified enzymatic quantitative PC-specific analysis showed that PC was the dominant PL in edible insects. In conclusion, edible insects, particularly migratory locusts and silkworms, may be novel sources of n-3 PUFAs containing functional PLs.

Анализ данных технохимического состава европейской химеры (Chimaera monstrosa) Северной Атлантики

Introduction. Deep-sea fauna of the North Atlantic has a great potential for the development of food industry. Rabbitfish (Chimaera monstrosa) is a hydrobionts that makes up a significant proportion of bycatch in the traditional trawl and longline fishery. The research objective was to determine the technochemical composition and biochemical properties of organs and tissues of rabbitfish. 
 Study objects and methods. The size-mass and total chemical composition was performed by standard methods. The amino acid composition of proteins was determined by chromatographic separation of amino acid derivatives obtained by reaction with orthophthalic aldehyde and β-mercaptoethanol. The method of one-dimensional thin-layer chromatography made it possible to determine the fractional composition of lipids. The fatty acid composition was determined using an S-180 gas-liquid chromatograph (Yanaco, Japan). The fat-soluble vitamins were determined by the saponification of samples with alkali, extraction, and separation of the unsaponifiable part. The fractional composition of proteins became clear after a planar polyacrylamide gel electrophoresis on (MultiPhor II, Sweden). 
 Results and discussion. The research featured the size-mass and chemical composition of body parts, the fractional and amino acid composition of proteins, as well as the fractional and fatty acid composition of lipids, vitamins, heavy metals, and organochlorine compounds in the tissues and organs of the rabbitfish. The sensory properties of the samples proved quite high. Hot smoked rabbitfish meat can be recommended for snack foods. Waste (heads, entrails, skin, cartilage, fins) makes up more than 50% of total body weight and can be used in feed production or as a protein-containing raw material for hydrolysates in microbiology, medicine, and food industry. 
 Conclusion. The research involved a complex biochemical study of the rabbitfish organs and tissues. The performed technochemical studies made it possible to give preliminary recommendations on the directions of its most rational integrated use.

Lipid Composition of the Tissues of Coregonus lavaretus L. of the Kola Peninsula

The article presents the results of the investigation of the lipid composition of the muscle, liver and eggs of the whitefish Coregonus lavaretus L. of different age groups harvested from lakes Lovozero and Umbozero of the Kola Peninsula. The study of the lipid composition of the tissues to some extent allows evaluating the features of lipid metabolism and the physiological state of an organism. Lipids were extracted from the tissues applying Bligh – Dyer method. Fractional composition of lipids was determined using one-dimensional thin layer chromatography. Individual lipid fractions were determined by densitometry. The content of total lipids in the muscle tissue of mature whitefish from Umbozero Lake was lower than that of the whitefish from Lovozero Lake. With ageing, the content of phospholipids in the muscle tissue of the whitefish from Umbozero unlike the whitefish from Lovozero increases, but the content of triacylglycerols decreases, due to the lack of nutrition and active swimming of the whitefish in cold water of Umbozero Lake. The portion of triacylglycerols in the liver of the whitefish from Lovozero is higher than in the whitefish from Umbozero, which is probably related to the diet of the whitefish from Lovozero. In the whitefish eggs from the two reservoirs structural lipids are dominant and high content of cholesterol was identified. Molar ratio of CHS/PHL and PHCH/PHEA is lower in the whitefish from Umbozero compared to the whitefish from Lovozero, which should lead to an increase in the functional activity of membrane structures, lipid matrix fluidity and metabolism in the fish from Umbozero.

Lipid-protein microdomains in tonoplast of Beta vulgaris L.: comparison between the results obtained by detergent and detergent-free isolation techniques

Abstract Objective The membranes of plant and animal cells contain lipid-protein microdomains (LPMs) in their structure. We aimed to compare biochemical and biophysical characteristics of LPMs isolated by the two techniques from beet root tonoplast. Methods For the purposes of comparison the biochemical characteristics of LPMs the content of protein (Bradford’s method), the content of lipids (gravimetric method) were assessed. For the purpose of separating different kinds of lipids, one-dimensional thin layer chromatography (TLC), two-dimensional TLC and different specific systems of solvents were used. The content of fatty acids and sterols was determined by chromatography-mass spectrometry and gas-liquid chromatography-mass spectrometry correspondingly. In order to determine the orderings of the lipids in LPMs the laser scanning confocal fluorescence microscopy was used. Results Significant differences were revealed as a result of investigation of biochemical and biophysical characteristics of LPMs. The total content of proteins was substantially higher (4 times as large) in LPMs identified by the detergent-free technique than in LPMs identified by the detergent technique. The orderings of lipids in LPMs isolated by detergent-free technique was substantially lower than that of LPMs isolated by detergent technique. Conclusion These results may suggest an existence of several types of LPMs in tonoplast.

Plasma phospholipid changes are associated with response to chemotherapy in non-Hodgkin lymphoma patients

Limited studies have been performed to associate abnormal phospholipid (PL) profile and disease activity in hematological malignancies, including non-Hodgkin lymphoma (NHL). The aim of his study was to evaluate the levels of plasma PL fractions in NHL patients, in response to chemotherapy. Forty non-treated patients with NHL and 25 healthy individuals were recruited. Blood samples from patients were taken before chemotherapy, after 3 cycles and after the end of the treatment, and PL fractions were resolved by one-dimensional thin-layer chromatography. To assess potential relationship between plasma PL profile and response to therapy, patients were divided according to clinical outcome in 3 groups: complete remission (CR), stable disease (SD) and progression (PG). Despite significant differences between NHL patients and healthy controls, no differences were found at baseline among patients divided according to clinical outcome. During and after chemotherapy important alterations in PL profile were observed. Levels of total PLs and all PL fractions decreased in patients with PG while in patients who responded to therapy (CR, SD) PLs significantly increased. Results of our study suggest that changes of total PLs and PL fractions during the therapy are associated with the effects of therapy and clinical outcome in patients with NHL.

Analysis of Plasmalogen Synthesis in Cultured Cells.

Plasmalogen synthesis can be analyzed by metabolic labeling, followed by the separation of ethanolamine plasmalogens from glycerophospholipids on one-dimensional thin-layer chromatography. The vinyl-ether bond of plasmalogens is acid-labile, which allows separating plasmalogens as 2-acyl-glycerophospholipids from diacyl-glycerophospholipids.

The New TLC Method for Separation and Determination of Multicomponent Mixtures of Plant Extracts.

The new mode of two-dimensional gradient thin layer chromatography (MGD-2D TLC) has been presented. Short distance development of sample in the first dimension leads to formation of the preconcentrated narrow zones. They are consecutively separated in the second dimension with the mobile phase gradient in several steps of development until the eluent reaches the further end of the chromatographic plate. The use of the above-mentioned technique allows isolating and then identifying the compounds of various polarity from the multicomponent mixture. The practical application of two-dimensional gradient thin layer chromatography has been performed for isolation of the two plant (Juniperus and Thymus) oils components as the examples of test mixtures. The experiments have been carried out with the use of silica gel plates as well as a normal phase condition. The results of solute separation with isocratic one-dimensional thin layer chromatography system have been compared with those of two-dimensional gradient system. It has been observed that application of the latter mode leads to almost triplicated number of zones in comparison with the former one. It is purposeful to apply the proposed mode to control the purity of the dominant component or components of the mixture.

The role of membrane lipids in forming the electrokinetic properties of red blood cells in solid tumors and psoriasis

Aim. Analysis of the electrophoretic mobility of red blood cells in patients with the diseases caused by increased cell proliferation activity, and its relationship with the lipid spectrum of erythrocyte membranes.
 Methods. The blood of 42 patients with malignant tumors of epithelial tissues (colon, kidney, prostate, urine bladder and cervical cancer) and 16 patients with skin diseases was studied. The control group consisted of 20 healthy individuals. Electrophoretic mobility of red blood cells was assessed by microelectrophoresis. Lipid fractions levels in erythrocytes was determined by one-dimensional thin layer chromatography.
 Results. Over than two-fold decrease of electrophoretic mobility of erythrocytes was observed in patients with malignant tumors of epithelial tissues and skin diseases associated with cell proliferation processes (psoriasis) - by 59 and 60%, respectively In inflammatory skin diseases (microbial eczema), the decrease was less pronounced (30%). The contents of charged phospholipid in erythrocyte membranes in case of cancer and psoriasis has changed in a similar way: a significant reduction in sphingomyelin level in the absence of significant differences in phosphatidylethanolamine and phosphatidylcholine levels. Multidirectional statistically significant changes in the contents of lysophosphatidylcholine were revealed depending on the stage of cancer, their level was decreased two-fold in case of psoriasis. The changes of cholesterol, diacylglycerols, free fatty acids levels in the membranes of red blood cells in case of cancer and psoriasis were divergent.
 Conclusion. In diseases caused by cell proliferation activation, electrophoretic mobility of erythrocytes is reduced by more than 50%. Kinetic characteristic of erythrocytes may be used for detecting the conditions of pathologic cell proliferation, including malignant tumors, since they alter in cases of phospholipid membranes lipid spectrum disorders.

The Influence of Image Correction and Chromatogram Alignment on Similarity Measure of One-Dimensional TLC by Means of Image Analysis

As a simple, rapid, low-cost method for densitometric evaluation of thin-layer chromatography (TLC), image analysis has increased in popularity in pattern recognition and fingerprint analysis in the context of TLC. Nonuniform image backgrounds and retention factor (Rf) variation are notable problems for the similarity measure of TLC by means of image analysis. In this work, their influences were studied. An image correction method and a chromatogram alignment method were designed. A Citrus herbal medicine was determined nine times on one plate and six times on independent plates separately. Mutual similarities of densitograms of replicate samples were measured using cosine measure and Pearson’s correlation measure. Paired-samples t-test was used to compare the means of the mutual similarities between two treatments. At last, a case study of classification of eight Citrus herbal medicines using hierarchical clustering was performed. The image correction and chromatogram alignment methods described in this work were proven effective. Image correction had a small influence on the similarity measure while chromatogram alignment had a great influence. Image correction and chromatogram alignment should be considered in the similarity measure of TLC by means of image analysis.

Membrane adaptation in phospholipids and cholesterol in the widely distributed, freeze-tolerant wood frog, Rana sylvatica

Maintaining proper membrane phase and fluidity is important for preserving membrane structure and function, and by altering membrane lipid composition many organisms can adapt to changing environmental conditions. We compared the phospholipid and cholesterol composition of liver and brain plasma membranes in the freeze-tolerant wood frog, Rana sylvatica, from southern Ohio and Interior Alaska during summer, fall, and winter. We also compared membranes from winter-acclimatized frogs from Ohio that were either acclimated to 0, 4, or 10 °C, or frozen to -2.5 °C and sampled before or after thawing. Lipids were extracted from isolated membranes, separated by one-dimensional thin-layer chromatography, and analyzed via densitometry. Liver membranes underwent seasonal changes in phospholipid composition and lipid ratios, including a winter increase in phosphatidylethanolamine, which serves to increase fluidity. However, whereas Ohioan frogs decreased phosphatidylcholine and increased sphingomyelin, Alaskan frogs only decreased phosphatidylserine, indicating that these phenotypes use different adaptive strategies to meet the functional needs of their membranes. Liver membranes showed no seasonal variation in cholesterol abundance, though membranes from Alaskan frogs contained relatively less cholesterol, consistent with the need for greater fluidity in a colder environment. No lipid changed seasonally in brain membranes in either population. In the thermal acclimation experiment, cold exposure induced an increase in phosphatidylethanolamine in liver membranes and a decrease in cholesterol in brain membranes. No changes occurred during freezing and thawing in membranes from either organ. Wood frogs use tissue-specific membrane adaptation of phospholipids and cholesterol to respond to changing environmental factors, particularly temperature, though not with freezing.

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

A simplified method for rapid quantification of intracellular nucleoside triphosphates by one-dimensional thin-layer chromatography

Quantification of nucleotides is an important part of metabolomics but has been hampered by the lack of fast, sensitive, and reliable methods. We present a less time-consuming, more sensitive, and more precise method for the quantitative determination of nucleoside triphosphates (NTPs), 5-ribosyl-1-pyrophosphate (PRPP), and inorganic pyrophosphate (PP i) in cell extracts. The method uses one-dimensional thin-layer chromatography (TLC) and radiolabeled biological samples. Nucleotides are resolved at the level of ionic charge in an optimized acidic ammonium formate and chloride solvent, permitting quantification of NTPs. The method is significantly simpler and faster than both current two-dimensional methods and high-performance liquid chromatography (HPLC)-based procedures, allowing a higher throughput while common sources of inaccuracies and technical problems are avoided. For determination of PP i, treatment with inorganic pyrophosphatase (PPase) of the radiolabeled phosphate is employed for removal of contaminating pyrophosphate. Biological examples performed in triplicates showed standard deviations of approximately 10% of the mean for the determined concentrations of NTPs.

Applied thin-layer chromatography: best practice and avoidance of mistakes

Applied thin-layer chromatography: best practice and avoidance of mistakes

Upregulated liver conversion of α-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet

We quantified incorporation rates of plasma-derived alpha-linolenic acid (alpha-LNA, 18:3n-3) into "stable" liver lipids and the conversion rate of alpha-LNA to docosahexaenoic acid (DHA, 22:6n-3) in male rats fed, after weaning, an n-3 PUFA-adequate diet (4.6% alpha-LNA, no DHA) or an n-3 PUFA-deficient diet (0.2% alpha-LNA, no DHA) for 15 weeks. Unanesthetized rats were infused intravenously with [1-14C]alpha-LNA, and arterial plasma was sampled until the liver was microwaved at 5 min. Unlabeled alpha-LNA and DHA concentrations in arterial plasma and liver were reduced >90% by deprivation, whereas unlabeled arachidonic acid (20:4n-6) and docosapentaenoic acid (22:5n-6) concentrations were increased. Deprivation did not change alpha-LNA incorporation coefficients into stable liver lipids but increased synthesis-incorporation coefficients of DHA from alpha-LNA by 6.6-, 8.4-, and 2.3-fold in triacylglycerol, phospholipid, and cholesteryl ester, respectively. Assuming that synthesized-incorporated DHA eventually would be secreted within lipoproteins, calculated liver DHA secretion rates equaled 2.19 and 0.82 micromol/day in the n-3 PUFA-adequate and -deprived rats, respectively. These rates exceed the published rates of brain DHA consumption by 6- and 10-fold, respectively, and should be sufficient to maintain normal and reduced brain DHA concentrations, respectively, in the two dietary conditions.

StarD10, a START Domain Protein Overexpressed in Breast Cancer, Functions as a Phospholipid Transfer Protein

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.

Separation of yeast phospholipids using one-dimensional thin-layer chromatography

Separation of yeast phospholipids using one-dimensional thin-layer chromatography

2D TLC — Graft planar chromatography in the analysis of a mixture of phenolic acids

Of the many chromatographic methods currently available, thin-layer chromatography (TLC) is widely used for the rapid analysis of plant extracts. Although, most practical planar chromatographic problems can be solved by use of a single TLC plate, the power of one-dimensional TLC is often inadequate for clean resolution of the compounds present in complex samples. A marked improvement of the separation for such samples can be achieved by multidimensional (MD) separation procedures. One possible form of multidimensional planar separation is the form of 2D TLC known as graft planar chromatography in which chromatographic plates are connected for capillary-controlled planar chromatography. In this variant of 2D TLC the first development can be performed on an RP-18W HPTLC plate and that in the perpendicular direction on a silica gel TLC plate.

Analysis of the products of mRNA decapping and 3'-to-5' decay by denaturing gel electrophoresis.

The majority of mRNA turnover is mediated either by mRNA decapping/5'-to-3' decay or exosome-mediated 3'-to-5' exonucleolytic decay. Current assays to assess mRNA decapping in vitro using cap-labeled RNA substrates rely on one-dimensional thin layer chromatography. This approach does not, however, resolve free phosphate from 7meGDP, the product of Dcp1p-mediated mRNA decapping. This can result in misinterpretation of the levels of mRNA decapping due to the generation of free phosphate following the action of the unrelated scavenger decapping activity on the products of exosome-mediated decay. In this report, we describe a simple denaturing acrylamide gel-based assay that faithfully resolves all of the possible products that can be generated from cap-labeled RNA substrates by turnover enzymes present in cell extracts. This approach allows a one-step assay to quantitatively assess the contributions of the exosome and DCP-1-type decapping on turnover of an RNA substrate in vitro. We have applied this assay to recalculate the effect of competition of cap-binding proteins on decapping in yeast. In addition, we have used the assay to confirm observations made on regulated mRNA decapping in mammalian extracts that contain much higher levels of exosome activity than yeast extracts.

Immunoaffinity column clean-up and thin layer chromatography for determination of ochratoxin A in green coffee

An immunoaffinity clean-up-based method for determining ochratoxin A (OTA) in green coffee aiming at one-dimensional thin layer chromatography (TLC) analysis was established. OTA was extracted with a mixture of methanol and aqueous sodium hydrogen carbonate solution, purified through an immunoaffinity column, separated on normal or reversed-phase (RP) TLC plates and detected and quantified by visual and densitometric analysis. The linear equation of the standard calibration curve by densitometric analysis gave R2 > 0.999 (0.04–84 ng). The mean recovery (R) of OTA from spiked samples (1.8–109 µg kg−1) by densitometric and visual analyses were 98.4 and 103.8%, respectively. The relative standard deviations (RSD) for densitometric and visual analysis varied from 1.1 to 24.9% and from 0.0 to 18.8%, respectively. The RSD for naturally contaminated samples by densitometry (three levels of contamination, n = 3) varied from 11.1 to 18.1%. The correlation (R2) between high-performance liquid chromatography (HPLC) and densitometry, and between visual and densitometric analysis for spiked samples were > 0.99. The limit of detection (LOD) of the method was 0.5 µg kg−1 for normal TLC. Toluene-ethyl acetate-88% formic acid (6:3:1 v/v/v) and acetonitrile-methanol-water-glacial acetic acid (35:35:29:10 v/v/v/v) were regarded as the suitable TLC solvents for eluting both standards and samples on normal and RP TLC plates, respectively. Toluene-acetic acid (99:1 v/v) was chosen as the spotting solvent among several others for giving the best sensitivity and resolution of OTA on TLC plates as well as the best recovery of OTA from standard and sample extract residues. Preliminary studies were carried out to investigate the reuse of the immunoaffinity column and the interference of caffeine in the OTA recovery.