Determination of aflatoxins B1, B2, G1 and G2 by high-performance liquid chromatography with electrochemical detection

A new approach to the determination of afiatoxins B₁, B₂, G₁ and G₂ is given; the method involves high-performance liquid chromatography with amperometric detection in the differential-pulse mode at the dropping mercury electrode with 1-s drop time. These aflatoxins can be determined simultaneously with good resolution but with some compromise in sensitivity. The detection limit of underivatized aflatoxin standards is around 5 ng. Average recoveries of aflatoxins from peanut butter by the Beebe method were G₂ 81%, G₁ 87%, B₂ 77% and B₁ 76%.     

Development of a microwave-assisted-extraction-based method for the determination of aflatoxins B1, G1, B2, and G2 in grains and grain products

This article describes the use of microwave-assisted extraction (MAE) as a pretreatment technique for the determination of aflatoxins B₁, G₁, B₂, and G₂ in grains and grain products. The optimal operation parameters, including extraction solvent, temperature, and time, were identified to be acetonitrile as the extraction solvent at 80 °C with 15 min of MAE. The extracts were cleaned up using solid-phase extraction followed by derivatization with trifluoroacetic acid and were determined by liquid chromatography–fluorescence detection. A Sep-Pak cartridge was chosen over Oasis HLB and Bond Elut cartridges. By the use of aflatoxin M₁ as an internal standard, relative recoveries of the aflatoxins ranged from 90.7 to 105.7 % for corn and from 88.1 to 103.4 % for wheat, with relative standard deviations between 2.5 and 8.7 %. A total of 36 samples from local markets were analyzed, and aflatoxin B₁ was found to be the predominant toxin, with concentrations ranging from 0.42 to 3.41 μg/kg.

Simultaneous determination of aflatoxin B1, B2, G1, and G2 in corn powder,edible oil,peanut butter,and soy sauce by liquid chromatography with tandem mass spectrometry utilizing turbulent flow chromatography

A novel fully automated method based on dual column switching using turbulent flow chromatography followed by liquid chromatography with tandem mass spectrometry was developed for the determination of aflatoxin B₁, B₂, G₁, and G₂ in corn powder, edible oil, peanut butter, and soy sauce samples. After ultrasound‐assisted extraction, samples were directly injected to the chromatographic system and the analytes were concentrated into the clean‐up loading column. Through purge switching, the analytes were transferred to the analytical column for subsequent detection by mass spectrometry. Different types of TurboFlow^TM columns, transfer flow rate, transfer time were optimized. The limits of detection and quantification of this method ranged between 0.2–2.0 and 0.5–4.0 μg/kg for aflatoxins in different matrixes, respectively. Recoveries of aflatoxins were in range of 83–108.1% for all samples, matrix effects were in range of 34.1–104.7%. The developed method has been successfully applied in the analysis of aflatoxin B₁, B₂, G₁, and G₂ in real samples.     

Simultaneous determination of aflatoxins B1, B2, G1, G2 in Fructus Bruceae by high‐performance liquid chromatography with online postcolumn photochemical derivatization

A simple, reliable, and highly sensitive method for the simultaneous determination of aflatoxin B₁, B₂, G₁, G₂ in Fructus Bruceae was developed using high‐performance liquid chromatography coupled to online postcolumn photochemical derivatization and fluorescence detection. Aflatoxins were first extracted by a methanol/water mixture and then cleaned up with an AflaTest? immunoaffinity column. Different clean‐up and derivatization methods were compared and optimized. The established method was extensively validated to show satisfactory performance of linearity (R² ≥ 0.9997), recovery (74.3–100.8%), and precision (RSDs ≤ 2.8%) for the investigated aflatoxins. This proposed method was also applied to 11 F. Bruceae samples and the results showed that 10 out of 11 were contaminated with aflatoxins ranging from 0.26 to 27.52 μg/kg and the occurrence of aflatoxin B₁, the most toxic one, was as high as 91% in all the samples, highlighting the severe contamination and the necessity to set legal limits for aflatoxins in F. Bruceae.     

Graphene‐coated magnetic‐sheet solid‐phase extraction followed by high‐performance liquid chromatography with fluorescence detection for the determination of aflatoxins B1, B2, G1, and G2 in soy‐based samples

A new method named graphene‐coated magnetic‐sheet solid‐phase extraction based on a magnetic three‐dimensional graphene sorbent was developed for the extraction of aflatoxins prior to high‐performance liquid chromatography with fluorescence detection. The use of a perforated magnetic‐sheet for fixing the magnetic nanoparticles is a new feature of the method. Hence, the adsorbent particles can be separated from sample solution without using an external magnetic field. This made the procedure very simple and easy to operate so that all steps of the extraction process (sample loading, washing, and desorption) were carried out continuously using two lab‐made syringe pumps. The factors affecting the performance of extraction procedure such as the extraction solvent, adsorbent dose, sample loading flow rate, ionic strength, pH, and desorption parameters were investigated and optimized. Under the optimal conditions, the obtained enrichment factors and limits of detection were in the range of 205–236 and 0.09–0.15 μg/kg, respectively. The relative standard deviations were <3.4 and 7.5% for the intraday (n = 6) and interday (n = 4) precisions, respectively. The developed method was successfully applied to determine aflatoxins B₁, B₂, G₁, and G₂ in different soy‐based food samples.     

Rapid Separation of Prostaglandins by Linear High Performance Thin Layer Chromatography

Abstract

A continuous development technique using silica gel linear high performance TLC plates is described for the separation of prostaglandins 6-keto-F_1α, F_2α, E₂, 13,14-dihydro-15-keto-F_2α, 13–14-dihydro-15-keto-E₂, and thromboxane B₂. Complete separation of all six prostaglandins was achieved with a solvent system of ethyl acetate/acetone/acetic acid (90:5:1). The method is simple, rapid and provides excellent resolution of plasma prostaglandins prior to quantitation by gas chromatography-mass spectrometry.     

Rapid analysis of aflatoxins B1, B2, and ochratoxin A in rice samples using dispersive liquid–liquid microextraction combined with HPLC

A novel, simple, and rapid method is presented for the analysis of aflatoxin B₁, aflatoxin B₂, and ochratoxin A in rice samples by dispersive liquid–liquid microextraction combined with LC and fluorescence detection. After extraction of the rice samples with a mixture of acetonitrile/water/acetic acid, mycotoxins were rapidly partitioned into a small volume of organic solvent (chloroform) by dispersive liquid–liquid microextraction. The three mycotoxins were simultaneously determined by LC with fluorescence detection after precolumn derivatization for aflatoxin B₁ and B₂. Parameters affecting both extraction and dispersive liquid–liquid microextraction procedures, including the extraction solvent, the type and volume of extractant, the volume of dispersive solvent, the addition of salt, the pH and the extraction time, were optimized. The optimized protocol provided an enrichment factor of approximately 1.25 and with detection of limits (0.06–0.5 μg/kg) below the maximum levels imposed by current regulations for aflatoxins and ochratoxin A. The mean recovery of three mycotoxins ranged from 82.9–112%, with a RSD less than 7.9% in all cases. The method was successfully applied to measure mycotoxins in commercial rice samples collected from local supermarkets in China.     

Distribution coefficients of vitamin B2 in hydrophilic organic solvent-aqueous salt solution systems

Distribution coefficients of vitamin B₂ in hydrophilic solvent (n-butanol, isopropanol, acetone, ethyl acetate, and their mixtures)-aqueous salt (potassium chloride, sodium fluoride, and ammonium sulfate salting-out agents) solution systems were calculated. The synergic effect and optimum proportions of components in the solvent mixture for efficient extraction of vitamin B₂ from aqueous solutions were established.     

Some interferences in radioimmunoassay of aflatoxin B1

The specificity of radioimmunoassay of aflatoxin B₁ was tested. Relative cross-reactivity of used antiserum with aflatoxins B₁, B₂, G₁, G₂ and M₁ was found to be 100%, 24%, 44.2%, 10.3%, and 1.4%, respectively. The interference of coumarin, albumins, steroids and ethylvanilin was estimated also in radioimmunoassay of aflatoxin B₁. Thus these compounds may cause a false positive finding of aflatoxin B₁.     

State‐state transitions for CCl2(X1A1, A1B1, a3B1) radical and collisional quenching of CCl2(A1B1 and a3B1) by O2, N2, NO,N2O,NH3, and various aminated molecules

CCl₂ free radicals were produced by a pulsed dc discharge of CCl₄ in Ar. Ground electronic state CCl₂(X) radicals were electronically excited to the A¹B₁ (0,4,0) vibronic state with an Nd:YAG laser pumped dye laser at 541.52 nm. Experimental quenching data of excited CCl₂(A¹B₁ and a³B₁) by O₂, N₂, NO, N₂O, NH₃, NH(CH₃)₂, NH(C₂H₅)₂, and N(C₂H₅)₃ molecules were obtained by observing the time‐resolved total fluorescence signal of the excited CCl₂ radical in a cell, which showed a superposition of two exponential decay components under the presence of quencher. The quenching rate constants k_A of CCl₂(A) state and k_a of CCl₂(a) state were derived by analyzing the experimental data according to a proposed three‐level model to deal with the CCl₂(X¹A₁, A¹B₁, a³B₁) system. The formation cross sections of complexes of electronically excited CCl₂ radicals with O₂, N₂, NO, N₂O, NH₃, and aminated molecules were calculated by means of a collision‐complex model. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 351–356, 2002     

One-Pot,Facile, Highly Efficient,and Green Synthesis of Acridinedione Derivatives Using Vitamin B1

An efficient methodology for the synthesis of acridinedione derivatives 4a–o has been achieved by one-pot, multicomponent condensation of dimedone 1, various amines 2a–d, and substitute aromatic aldehydes 3a–k, in the presence of the easily available, inexpensive, and nontoxic catalyst vitamin B₁ (VB₁) as a versatile biodegradable. Synthesis of acridine-type compounds was performed in good yields in water as green solvent. Its high-yield efficiency; clean, ecofriendly, simple workup procedure; and easy purification are regarded as the main advantages of this method besides its green solvent. The synthesized compounds are characterized using spectroscopic analyses (FTIR, ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry) techniques.     

Resolution of PTH-amino Acids in Three New Solvents on Zn,Cd, Hg Impregnated Silica Plates

Abstract

A ten component mixture of PTH-amino acids has been resolved by TLC using impregnated silica plates. Three new solvent systems, CHCl₃ -H₂O-EtOAc(28:1:1), CCl₄-AcOH(19:1) and CHC₃-MeOH-Benzene(19:1:5) were developed and used. The effect of 0.1, 0.2 and 0.5 % concentrations of each of the impregnant (Zn⁺⁺, Cd⁺⁺, Hg⁺⁺ ions) on R_F values was studied.     

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.     

Colloidal gold based immunochromatographic strip for the simple and sensitive determination of aflatoxin B1 and B2 in corn and rice

We have developed a simple and fast immunochromatographic test strip for the simultaneous quantitation of aflatoxin B₁ and aflatoxin B₂ in corn and rice. The strip contains three pads (sample, conjugate, and absorbing pad) and uses the respective polyclonal antibodies immobilized on gold nanoparticles. Matrix interferences were minimized by application of fugacity theory. Clean-up of samples and pre-treatment of strip pads is not required. The visual detection limit is 0.1 ng mL^?1, and the process can be completed within 5 min. Out of 113 natural samples, 16 rice and 27 corn samples (38% in total) were aflatoxin positive and the test results were confirmed by HPLC. The strip shows, however, high cross reactivity to aflatoxins G₁, G₂, and M₁. We consider this strip to possess wide applicability because of its ease of use, sensitivity, stability, and low cost.

Application of molecularly imprinted polymers to determine B1, B2, and B3 fumonisins in cereal products

The aim of this study was to develop an analytical method for qualitative and quantitative determination of the B₁, B_2, and B₃ fumonisins in cereal products. A LC coupled to an IT‐MS was used as the analytical instrument. The AFFINIMIP FumoZON Molecularly Imprinted Polymer SPE cartridges (Polyintell) were used to isolate fumonisins from the analyzed samples and the clean‐up step. Statistical parameters evaluated in some validation experiments were as follows: mean recovery 95–106%, precision <17% (expressed as recovery RSD). The developed method was used to determine fumonisins in 49 cereals (42 maize‐based and seven wheat‐based products). In most cases, concentrations of the studied compounds found in the analyzed samples were low. The highest total concentration of the B₁, B₂, and B₃ fumonisins was found in maize flour samples (range, 26–1102 μg/kg, mean 498 μg/kg).     

Separation of Prostaglandins E1, and E2 and other Major Eicosanoids by Unidimensional Argentation Thin Layer Chromatography

Abstract

Prostaglandins (PG) E₁ and E₂ are important regulators of biologic functions, and can express different biological effects. In thin layer chromatography (TLC) systems which separate these compounds, comigration of other major eicosanoids is a problem. This paper describes a TLC system using a mobile phase of chloroform/methanol/acetic acid/H₂O (90:7.5:5:0.8) that separates PGE₁ and PGE₂, as well as other major eicosanoids, including dihomogammalinolenic acid (DHLA), the immediate fatty acid precursor of PGE₁.     

Thin Layer Chromatographic Separation of Thorium,Titanium, Zirconium and Some Rare Earths on Cellulose Impregnated Layers

Abstract

The chromatographic saparation of Thorium, Titanium, Zirconium and eight rare earths has been attempted on DBBP, HDEHP and HDEHP-TOPO impregnated cellulose plates using the solvent system MaOH-6N HNO₃?Acatyl acetone (20:15:5). An efficient seperation of 4–5 rare earths on HDEHP-TOPO impregnated plates has been achieved.     

Syntheses and Crystal Structures of Rb2B2Se7, Tl2B2Se7, Cs3B3Se10, and Tl3B3Se10: New Perseleno‐selenoborates with Polymeric Anionic Chains

The perseleno‐selenoborates Rb₂B₂Se₇ and Cs₃B₃Se₁₀ were prepared from the metal selenides, amorphous boron and selenium, the thallium perseleno‐selenoborates Tl₂B₂Se₇ and Tl₃B₃Se₁₀ directly from the elements in evacuated carbon coated silica tubes by solid state reactions at temperatures between 920 K and 950 K. All structures were refined from single crystal X‐ray diffraction data. The isotypic perseleno‐selenoborates Rb₂B₂Se₇ and Tl₂B₂Se₇ crystallize in the monoclinic space group I 2/a (No. 15) with lattice parameters a = 12.414(3) Å, b = 7.314(2) Å, c = 14.092(3) Å, β = 107.30(3)°, and Z = 4 for Rb₂B₂Se₇ and a = 11.878(2) Å, b = 7.091(2) Å, c = 13.998(3) Å, β = 108.37(3)° with Z = 4 for Tl₂B₂Se₇. The isotypic perseleno‐selenoborates Cs₃B₃Se₁₀ and Tl₃B₃Se₁₀ crystallize in the triclinic space group P1 (Cs₃B₃Se₁₀: a = 7.583(2) Å, b = 8.464(2) Å, c = 15.276(3) Å, α = 107.03(3)°, β = 89.29(3)°, γ = 101.19(3)°, Z = 2, (non‐conventional setting); Tl₃B₃Se₁₀: a = 7.099(2) Å, b = 8.072(2) Å, c = 14.545(3) Å, α = 105.24(3)°, β = 95.82(3)°, γ = 92.79(3)°, and Z = 2). All crystal structures contain polymeric anionic chains of composition ([B₂Se₇]^2–)_n or ([B₃Se₁₀]^3–)_n formed by spirocyclically fused non‐planar five‐membered B₂Se₃ rings and six‐membered B₂Se₄ rings in a molar ratio of 1 : 1 or 2 : 1, respectively. All boron atoms have tetrahedral coordination with corner‐sharing BSe₄ tetrahedra additionally connected via Se–Se bridges. The cations are situated between three polymeric anionic chains leading to a nine‐fold coordination of the rubidium and thallium cations by selenium in M₂B₂Se₇ (M = Rb, Tl). Coordination numbers of Cs⁺ (Tl⁺) in Cs₃B₃Se₁₀ (Tl₃B₃Se₁₀) are 12(11) and 11(9).     

Na2B2Se7, K2B2S7 und K2B2Se7: Drei Perchalkogenoborate mit neuem polymeren Anionengerüst

Na₂B₂Se₇, K₂B₂S₇, and K₂B₂Se₇: Three Perchalcogenoborates with a Novel Polymeric Anion Network Na₂B₂Se₇ (I 2/a; a = 11.863(4) Å, b = 6.703(2) Å, c = 13.811(6) Å, β = 109.41(2)°; Z = 4), K₂B₂S₇ (I 2/a; a = 11.660(2) Å, β = 6.827(1) Å, c = 12.992(3) Å, β = 106.78(3)°; Z = 4), and K₂B₂Se₇ (I 2/a; a = 12.092(4) Å, b = 7.054(2) Å, c = 13.991(5) Å, β = 107.79(3)°; Z = 4) were prepared by reaction of stoichiometric amounts of sodium selenide (potassium sulfide) with boron and sulfur or of potassium selenide and boron diselenide, respectively, at 600°C with subsequent annealing. The crystal structures consist of polymeric anion chains of composition ([B₂S₇]^2?)_n or ([B₂Se₇]^2?)_n formed by spirocyclically connected five-membered B₂S₃ (B₂Se₃) rings and six-membered B₂S₄ (B₂Se₄) rings. The nine-coordinate alkaline metal cations are situated in between.     

Fertigs?ulen-Extraktion und quantitative HPTLC-Bestimmung der Aflatoxine B1, B2, G1 und G2 in Pilzn?hrl?sungen

Zusammenfassung Ausgehend von reinen Aflatoxin-Standardlösungen werden die Optimierung des dünnschicht-chromatographischen Trennschrittes sowie die Fluorescenzstabilisierung durch spezielle postchromatographische Behandlung der HPTLC-Platten beschrieben. Die quantitative Bestimmung der Aflatoxine B₁; B₂, G₁ und G₂ in biologischer Matrix (Eurotium-repens-Pilznährlösungen) mittels der quantitativen HPTLC ist jedoch nur nach vorheriger Extraktion möglich. Die Fertigsäulenextraktion wird hinsichtlich Präzision und Richtigkeit mit der klassischen Flüssig/Flüssig-Extraktion im Scheidetrichter verglichen. Systematische Untersuchungen wäßriger Eichlösungen und aufgestockter Nährbodenlösungen zeigen die hohe Wiederfindungsrate und ausgezeichnete Präzision des Fertigsäulen-Extraktionsverfahrens. Aufstockexperimente von Pilznährlösungen (Eurotium repens) ergeben Wiederfindungsraten zwischen 96% und 101%. Die hier vorgestellten Ergebnisse zeigen, daß dieses quantitative HPTLC-Verfahren für die Routinebestimmung der Aflatoxine gut geeignet ist.

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Pre-packed-column extraction and quantative HPTLC-determinationof the aflatoxins B₁, B₂, G₁ and G₂ in fungal suspensions

Summary The method for the quantitative determination of aflatoxin in fungal suspensions and in stock solutions was improved. The thin-layer chromatographic separation was optimized and the fluorescence intensity was stabilized and enhanced by a special post-chromatographic treatment of the HPTLC-plates.The quantitative determination of aflatoxin B₁; B₂, G₁ and G₂ in biological matrix (Eurotium repens) has been proved to be only possible after previous extraction. The pre-packed column extraction is compared with the classic liquid/liquid extraction with regard to precision and accuracy.Systematic investigations of aqueous standard and mold medium reveal high recovery rates and excellent accuracy of the pre-packed column extraction method. Recovery rates from 96% to 101% were obtained for experiments with standard-addition to fungal suspensions.The presented results indicate that the quantitative HPTLC-procedure is appropriate for routine analysis of aflatoxins.

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Diese Arbeit ist Teil der Dissertation von Frau G. Chalela im Fachbereich 15 (Institut für Pflanzenökologie) der Justus-Liebig-Universität Gießen

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Diese Arbeit wurde mit freundlicher Unterstützung der Firmen E. Merck (Darmstadt) und Camag (Muttenz, Schweiz) durchgeführt.