An Improved Method to Resolve Plant Saponins and Sugars by TLC

Thin-layer chromatography (TLC) plays an important role in the initial selection of mutants having a unique seed saponin composition from the germplasm collections of the subgenus Soja. In the conventional TLC procedure, the dehydrated free sugars are retained just below the major saponins and interrupt the identification of some minor saponin constituents. To resolve this problem, we developed an efficient and reliable method to move sugars from the saponin area on TLC. A developing chamber was saturated with the lower phase of chloroform:methanol:water (65:35:10, v/v) for 2 h and the TLC plates were developed in it for 50 min. Plates were then dried at 100 °C for 10 min to evaporate the excess mobile phase and developed again with 10 % H₂SO₄ for 15 min. While sulfuric acid migrates over the surface of SiO₂, sugar molecules are dehydrated and hydrophilic interactions between free sugars and SiO₂ are strongly reduced. Thus, the positions of dehydrated sugars were shifted to above the saponin area on the TLC plate. This resulted in easy recognition of the saponin composition without any discrimination. This amended protocol would be applicable to all TLC analyses in which the target components should be separate from the interrupting sugar molecules.

     

Semi-Preparative High Performance Liquid Chromatographic Separation of Carbon-14 Labeled Avermectin B1a from a Mixture of Avermectins

Abstract

A semi-preparative high performance liquid chromatographic method has been developed to separate carbon-14 labeled avermectin B_1a from a fermentation mixture of carbon-14 labeled avermectins, i. e., avermectins A₁a, A₁b, A₂a, A₂b, B₁a, B₁b, B₂a, and B₂b. Two HPLC systems were employed for the separation: I. A Whatman M20, Partisil 10, normal phase column and a solvent system of 10% ethanol in isooctane (v/v), and II. A Whatman M20, Partisil 10, ODS-3, reverse phase column and a solvent system of acetonitrile/methanol/water (56:18:26, v/v/v); the flow rate was 18 ml/ min. Avermectin separations were monitored using ultraviolet detection (254 nm). Further analyses of avermectin B₁a were done using analytical HPLC and TLC/radioassay to check compound purity and identity.     

Application of high‐performance countercurrent chromatography for the isolation of steroidal saponins from Liriope plathyphylla

High‐performance countercurrent chromatography (HPCCC) with electrospray light‐scattering detection was applied for the first time to isolate a spirostanol and a novel furostanol saponin from Liriope platyphylla. Due to the large differences in K_D values between the two compounds, a two‐step HPCCC method was applied in this study. The primary HPCCC employed methylene chloride/methanol/isopropanol/water (9:6:1:4 v/v, 4 mL/min, normal‐phase mode) conditions to yield a spirostanol saponin ( 1 ). After the primary HPCCC run, the solute retained in the stationary phase (SP extract) in HPCCC column was recovered and subjected to the second HPCCC on the n‐hexane/n‐butanol/water system (1:9:10 v/v, 5 mL/min, reversed‐phase mode) to yield a novel furostanol saponin ( 2 ). The isolated spirostanol saponin was determined to be 25(S)‐ruscogenin 1‐O‐β‐d ‐glucopyranosyl (1→2)‐[β‐d ‐xylopyranosyl (1→3)]‐β‐d ‐fucopyranoside (spicatoside A), and the novel furostanol saponin was elucidated to be 26‐O‐β‐d ‐glucopyranosyl‐25(S)‐furost‐5(6)‐ene‐1β‐3β‐22α‐26‐tetraol‐1‐O‐β‐d ‐glucopyranosyl (1→2)‐[β‐d ‐xylopyranosyl‐(1→3)]‐β‐d ‐fucopyranoside (spicatoside D).     

The effect of storage temperature on interactions between dehydrated sugars and phosphatidylcholine

We studied the effects of storage temperature on the stability of dehydrated POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine) mixed with sucrose, trehalose, or a sucrose/raffinose mixture. We used DSC to measure the gel-to-fluid phase transition temperature (T _m) of POPC after incubation either below or near the glass transition temperature (T _g) of the sugars in the mixture. Glass formation by the sugars around fluid-phase POPC led to the lowering ofT _m below that of the fully hydrated lipid. Phospholipid phase behavior did not change during storage belowT _g. In some samples stored aboveT _g, trehalose crystallized completely; in these samples, theT _g of POPC increased to that of the partially dehydrated phospholipid. Melting the crystalline sugar re-established its ability to lower POPC'sT _m. We conclude that prevention of complete sugar crystallization was important for stability in the dry state, and that storage belowT _g conferred long-term stability to the dehydrated sugar-lipid mixtures.     

Comparative potentiality of Kans grass (Saccharum spontaneum) and Giant reed (Arundo donax) as lignocellulosic feedstocks for the release of monomeric sugars by microwave/chemical pretreatment

Two-stage microwave (microwave/NaOH pretreatment followed by microwave/H₂SO₄ pretreatment) was used to release monomeric sugars from Kans grass (Saccharum spontaneum) and Giant reed (Arundo donax). The optimum pretreatment conditions were investigated, and the maximum monomeric sugar yields were compared. The microwave-assisted NaOH and H₂SO₄ pretreatments with a 15:1 liquid-to-solid ratio were studied by varying the chemical concentration, reaction temperature, and reaction time to optimize the amount of monomeric sugars. The maximum amounts of monomeric sugars released from microwave-assisted NaOH pretreatment were 6.8 g/100 g of biomass [at 80 °C/5 min, 5 % (w/v) NaOH for S. spontaneum and at 120 °C/5 min, 5 % (w/v) NaOH for A. donax]. Furthermore, the maximum amounts of monomeric sugars released from microwave-assisted H₂SO₄ pretreatment of S. spontaneum and A. donax were 33.8 [at 200 °C/10 min, 0.5 % (w/v) H₂SO₄] and 31.9 [at 180 °C/30 min, 0.5 % (w/v) H₂SO₄] g/100 g of biomass, respectively. The structural changes of S. spontaneum and A. donax were characterized using Fourier transform infrared spectroscopy and scanning electron microscopy.     

Synthesis and characterization of FePt nanoparticles and FePt nanoparticle/SiO2-matrix composite films

Superparamagnetic face-centered cubic (fcc) FePt nanoparticles were synthesized using a polyol process. The effect of reaction temperature and molar ratio of Fe(CO)₅ to Pt(acac)₂ on the structure, composition and morphology of nanoparticles has been investigated. The optimum processing condition has been obtained for producing well-monodisperse fcc-phase FePt nanoparticles with the 2:1?molar ratio of Fe-Pt at 220?°C. In order to circumvent the problem of FePt particle coalescence during high temperature annealing for the L1₀ ordering, FePt nanoparticle/SiO₂-matrix composite films have been fabricated by sol?Cgel method. The experimental results confirm that the amorphous SiO₂ matrix effectively inhibits the grain growth and particle aggregation during 700?°C annealing for 1?h. Well-monodisperse face-centered tetragonal (fct) FePt particles embedded in the SiO₂ matrix can be obtained with the long-range chemical order parameter S of ~0.74, indicating partially ordered L1₀ phase transition in FePt/SiO₂ composite films. The FePt/SiO₂ system exhibits a hysteretic behavior with smaller coercive field of 1,450 Oe. The incomplete phase transition from cubic deredat height maxsium (A ₁-disordered phase to tetragonal L1₀-ordered phase) might be responsible for it.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

Determination of Total Radiochemical Purity of [18F]FDG and [18F]FET by High-Performance Liquid Chromatography Avoiding TLC Method

The goal of this work was to present two high-performance liquid chromatography (HPLC) method that could be applied for the determination of the total radioactive purity of 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) and O-(2-[¹⁸F]fluoroethyl)-L-tyrosine ([¹⁸F]FET). The separation of [¹⁸F]fluoride ions, [¹⁸F]FET and [¹⁸F]FET intermediate was accomplished on LiChrosper RP-18, 250?×?4 mm, 5 µm (Merck) analytical column. For mobile phase 10 mM potassium dihydrogen phosphate buffer at pH7 (A) and acetonitrile (B) was used: 0–2 min: 15% B; 2–12 min: 85% B; 12–15 min: 15% B, respectively. Analysis of [¹⁸F]FDG was performed using LiChrosper 100 NH₂, 250?×?4.5 mm, 5 µm (Merck) analytical column. The initial mobile phase composition was 10 mM KH₂PO₄ buffer (pH7) and acetonitrile (15:85, v/v) and the acetonitrile ratio was decreased to 15% at 2 min after the sample injection and held for 5 min. Complete elution of [¹⁸F]fluoride ions from stationary phases could be achieved by adding 10 mg/mL K[¹⁹F]F to radioactive samples in a ratio 1:1 during the sample preparation. Recovery of [¹⁸F]fluoride ions ranged from 99.5 to 100.6%. The validation of the developed methods showed good results for linearity (r²?=?0.9981–0.9996), specificity (R_S?=?3.7–10.2), repeatability (%Area RSD_%?=?1.2–4.3%) and limit of quantitation (LOQ?=?1.6–4.5 kBq). During the cross-validation similar radiochemical purity values were obtained by the novel HPLC methods and thin layer chromatography performed according to the recommendations of the Ph. Eur. monographs.

     

Determination of Selected Quinolones and Fluoroquinolones by Use of TLC

Abstract

There are many different methods of quinolones determination. The most often used method of quinolones analysis is liquid chromatography. In this work some selected quinolones (cinoxacin, pipemidic acid) and fluoroquinolones (ofloxacin, pefloxacin) were separated with thin-layer chromatography (TLC). The two different mobile phases were used as follows: buffer solution (pH = 5.5)-methanol, 40:10 (v/v) and acetonitrile-water-acetic acid, 6:40:4 (v/v/v), respectively, for quinolones and fluoroquinolones. The following chromatographic parameters were calculated for these separations: R_F, ?R_F, R_M, and R_S. The possibility of qualitative determination of cinoxacin, pipemidic acid, ofloxacin, and pefloxacin using TLC was shown.     

Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min⁻¹ for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.

     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min^?1 for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.     

Catalysts based on cordierite modified with transition metal oxides

Catalysts active in ammonia oxidation have been obtained by the substitution of transition metal (Mn, Fe, Co, Ni, and Cu) ions for Mg ions in the cordierite structure 2MgO · 2Al₂O₃ · 5SiO₂ at 1100°C. Their phase composition, texture, and activity depend on the type and amount of introduced transition metal oxide. The Mn- and Cu-containing catalysts, which consist of substituted cordierites 2(Mg_{1 ? x} M _x )O · 2Al₂O₃ · 5SiO₂ and Mn₂O₃ or CuO crystallites located on their surface, are most active in ammonia oxidation. The catalysts are characterized by a small specific surface area and have large pores, whose total volume is small. The Fe-containing catalysts consist of the Fe-substituted cordierite phase and particles of an iron oxide phase. These particles are mostly located in internal pores of the catalysts and are, therefore, hardly accessible to ammonia molecules. The introduction of Co or Ni oxide leads to the formation of a low-active spinel phase rather than the cordierite phase.     

Untersuchungen an Nickeloxid-Mischkatalysatoren. XVI. Reduktionsverhalten amorpher NiO?Al2O3/SiO2-Katalysatoren

Studies on Nickel Oxide Mixed Catalysts. XVI. Reduction Behaviour of Amorphous NiO? Al₂O₃/SiO₂ Catalysts The reduction behaviour of NiO? Al₂O₃/SiO₂ catalysts prepared by precipitationdeposition is influenced by the phase composition (amorphous nickel layersilicates and nickel alumino layersilicates, nickel spinels, nickel oxide) and the differences of the composition between surface and bulk. TPR measurements, determinations of the reduction degree, and the nickel particle sizes by static magnetic measurements showed that the reducibility of the NiO? Al₂O₃/SiO₂ catalysts is enhanced and the nickel dispersity is decreased at low Al₂O₃ contents. The decrease of the reducibility at Al₂O₃ contents >5 mole% is caused by the formation of nickel spinels and the decrease of the Ni^II ion surface concentration.     

Effects of the ratio of Fe to Co over Fe-CO/SiO2 bimetallic catalysts on their catalytic performance for Fischer-Tropsch synthesis

The Fe-Co/SiO2 bimetallic catalysts with different ratios of Fe to Co were prepared by aqueous incipient wetness impregnation. The catalysts of 10%Fe:0%Co/SiO2, 10%Fe:6%Co/SiO2, 10%Fe:2%Co/SiO2, 10%Fe:10%Co/SiO2, 6%Fe:10%Co/SiO2, 2%Fe:10%Co/SiO2 and 0%Fe: 10%Co/SiO2 by mass were tested in a fixed reactor by the Fischer-Tropsch synthesis. Activity and hydrocarbon distribution were found to be determined by the ratio of iron to cobalt of the catalysts. Higher iron content inhibited the activity, whereas higher cobalt content enhanced the activity of the Fe:Co/SiO2 catalysts. On the other hand, for the catalysts of 10%Fe:6%Co/SiO2, 10%Fe:10%Co/SiO2, 6%Fe:10%Co/SiO2, and 2%Fe:10%Co/SiO2, the total C2–C4 fraction increased (from 10.65% to 26.78%) and C5+ fraction decreased (from 75.75% to 57.63%) at 523 K. Temperature programmed reduction revealed that the addition of cobalt enhanced the reducibility of the Fe:Co/SiO2 catalyst. Metal oxides were present in those catalysts as shown by XRD. The Fe-Co alloy phase was found in the 2%Fe:10%Co/SiO2, 6%Fe:10%Co/SiO2, 10%Fe:10%Co/SiO2, 10%Fe:6%Co/SiO2 catalysts and their crystals were perfect.     

Different stability‐indicating chromatographic methods for specific determination of paracetamol,dantrolene sodium,their toxic impurities and degradation products

A well‐known analgesic (paracetamol, PAR) and skeletal muscle relaxant [dantrolene sodium (DNS)] have been analyzed without interference from their toxic impurities and degradation products. The studied PAR impurities are the genotoxic and nephrotoxic p‐amino phenol (PAP) and the hepatotoxic and nephrotoxic chloroacetanilide, while 5‐(4‐nitrophenyl)‐2‐furaldehyde is reported to be a mutagenic and carcinogenic degradation product of DNS. The five studied components were determined and quantified by TLC–densitometric and RP‐HPLC methods. TLC–densitometry (method 1) used TLC silica gel and chloroform–ethyl acetate–acetic acid–triethylamine (7:3:0.5:0.05, by volume) as the mobile phase with UV scanning at 230 nm, while RP‐HPLC (method 2) was based on separation on a C₁₈ column using methanol–water (55:45, v/v pH 3 with aqueous formic acid) as mobile phase at 1 mL/min and detection at 230 nm. The developed methods were used for determination and quantification of the five studied components in different laboratory‐prepared mixtures. The were also applied for analysis of Dantrelax^® compound capsules where no interference among the studied components with each other or from excipients was observed. The methods were validated as per International Conference on Harmonization guidelines, and they compared favorably with the reported ones.     

Stereo‐ and Regioselective Direct Multi‐Deuterium‐Labeling Methods for Sugars

Deuterium‐labeled sugars can be utilized as powerful tools for the architectural analyses of high‐sugar‐containing molecules represented by the nucleic acids and glycoproteins, and chiral building blocks for the syntheses of new drug candidates (heavy drugs) due to their potential characteristics, such as simplifying the ¹H NMR spectra and the stability of C? D bonds compared with C? H bonds. We have established a direct and efficient synthetic method of deuterated sugars from non‐labeled sugars by using the heterogeneous Ru/C‐catalyzed H–D exchange reaction in D₂O under a hydrogen atmosphere with perfect chemo‐ and stereoselectivities. The direct H–D exchange reaction can selectively proceed on carbons adjacent to the free hydroxyl groups, and the deuterium labeling of various pyranosides (such as glucose and disaccharides), as well as furanosides, represented by ribose and deoxyribose was realized. Furthermore, the desired number of deuterium atoms can be freely incorporated into selected positions by the site‐selective protection of the hydroxyl groups using acetal‐type protective groups because the deuterium exchange reaction never proceeds on positions adjacent to the protected hydroxyl groups.     

Solvent extraction of uranium from lean grade acidic sulfate leach liquor with alamine 336 reagent

This paper describes the solvent extraction studies carried out on an acidic low assay uranium bearing leach liquor generated during sulfuric acid leaching of a refractory uranium ore using alamine 336?Cisodecenol?Ckerosene reagent combine. The leach liquor has a U₃O₈ content of about 270?mg/L, free acidity 2.4?N H₂SO₄ and total dissolved solids concentration of 260?g/L. Process parameteric variation studies indicated strong influence of free acidity of the leach liquor, alamine 336 concentration and aqueous to organic phase ratio on the extraction efficiency of uranium. An extraction efficiency of about 95% was achieved when the free acidity of leach liquor was 1?N H₂SO₄ or lower, using 2% (v/v) alamine 336 at ambient temperature with an aqueous to organic phase ratio of 1:1. The loading capacity under these conditions was 1.2?g/L of U₃O₈. About 98% of the uranium values could be stripped from the loaded organic using 1?N NaCl in 0.2?N H₂SO₄. The solvent extraction studies aided in developing a suitable process flowsheet for treating refractory uranium ores which need high acidity during leaching and relatively lower acidity for purification by solvent extraction.     

Effect of SiO<Subscript>2</Subscript> on the physicochemical and catalytic properties of VMoTeNbО catalyst in oxidative conversion of ethane

Supported oxide catalysts of the overall composition V_0.3Mo₁Te_0.23Nb_0.12/n SiO₂ (n = 0, 10, 25, 35, and 50 wt %) were tested in oxidative conversion of ethane to ethylene and were characterized by chemical analysis, X-ray diffraction, and high-resolution transmission electron microscopy. On introducing SiO₂, coarse crystals of the active М1 phase become partially coated with layers of amorphous SiO₂. The support does not influence the selectivity with respect to the reaction products. The catalysts with 10–25 wt % SiO₂ content exhibit the highest activity owing to the presence of nanodomains of the M1 phase.     

Development of an HPTLC‐based diagnostic method for invasive aspergillosis

A rapid, sensitive and specific high‐performance thin‐layer chromatographic (HPTLC) method was developed and validated for determination of gliotoxin in Aspergillus infected immunocompromised patients with invasive aspergillosis (IA). Densitometric analysis of gliotoxin was carried out in the absorbance mode at 254 nm after single‐step extraction with chloroform. The method uses TLC aluminum plates pre‐coated with silica gel 60F‐254 as a stationary phase and toluene–isoamyl alcohol–methanol (10:0.5:0.5, v/v/v) as mobile phase, which gives compact spot of gliotoxin (R_f = 0.51). The calibration curve was linear (r² ≥ 0.994) between peak area and concentration in the tested range of 100–1000 ng spot^?1 with minimum detectable range 0.025 ng μ^?1 of serum sample. The mean ± SD value of slope and intercept of the standard chromatogram of gliotoxin were found to be 523.2 ± 1.555635 and 915.8 ± 30.68843, respectively. The developed method is simple, rapid, precise and less costly than earlier diagnostic methods, and different serum samples can be run on a single TLC plate for comparative analysis. The proposed method can be used to analyze gliotoxin in patient serum for easy, rapid and cost‐effective diagnosis of IA. Copyright © 2009 John Wiley & Sons, Ltd.