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.     

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.     

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%.     

Simultaneous determination of four aflatoxins and ochratoxin A in ginger and related products by HPLC with fluorescence detection after immunoaffinity column clean‐up and postcolumn photochemical derivatization

Ginger, a widely used spice and traditional Chinese medicine, is prone to be contaminated by mycotoxins. A simple, sensitive, and reproducible method based on immunoaffinity column clean‐up coupled with HPLC and on‐line postcolumn photochemical derivatization with fluorescence detection was developed for the simultaneous determination of aflatoxins (AFs) B₁, B₂, G₁, G₂, and ochratoxin A (OTA) in 25 batches of gingers and related products marketed in China for the first time. The samples were first extracted by ultrasonication with methanol/water (80:20, v/v) and then cleaned up with immunoaffinity columns for analysis. Under the optimized conditions, the LODs and LOQs for the five mycotoxins were 0.03–0.3 and 0.1–0.9 μg/kg, respectively. The average recoveries ranged from 81.3–100.8% for AFs and from 88.6–99.5% for OTA at three spiking levels. Good linearity was observed for the analytes with correlation coefficients all >0.9995. All moldy gingers were contaminated with at least one kind of the five investigated mycotoxins, while none of them were found in normal gingers. Ginger powder samples were contaminated slightly with the contamination levels below the LOQs, while ginger tea bags were mainly contaminated by OTA at 1.05–1.19 μg/kg and ginger black tea bags were mainly contaminated by AFs at 3.37–5.76 μg/kg. All the contamination levels were below the legally allowable limits.     

Determination of Aflatoxins in Rice and Maize by Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry with Accelerated Solvent Extraction and Solid-Phase Extraction

A fast and reliable ultra-high performance liquid chromatography–tandem mass spectrometry method was developed for the determination of aflatoxins B₁, B₂, G₁, and G₂ in cereal. The analytes were extracted by accelerated solvent extraction with methanol/water (80:20). A polymeric solid-phase extraction column was used for sample preparation. Under optimum conditions, the analyte recoveries for samples spiked at different concentration levels in rice and maize ranged from 71.2 to 94.0%, with relative standard deviations less than 16.4%. Limits of detection (signal-to-noise ratio, 3:1) for the aflatoxins ranged from 0.25 to 0.93 ng/g. The developed method was applied to the determination of aflatoxins in ten rice and maize samples. One maize sample tested positive with an aflatoxin B₁ concentration of 2.7 ng/g.     

Extraction of aflatoxins from food samples using graphene‐based magnetic nanosorbents followed by high‐performance liquid chromatography: A simple solution to overcome the problems of immunoaffinity columns

In this research, magnetic graphene nanoparticles were prepared and used as adsorbents for preconcentrating the aflatoxins in rice, wheat, and sesame samples. For this purpose, graphene was synthesized by Hummer's method. Magnetically modified graphene formed by the deposition of magnetite (Fe₃O₄) on graphene was used for the separation of aflatoxins B₁, B₂, G₁, and G₂ from the samples. The extractants were subsequently analyzed with high‐performance liquid chromatography and fluorescence detection. Parameters affecting the efficiency of the method were thoroughly investigated. The measurements were done under the optimized conditions. For aflatoxins B₁, B₂, G₁, and G₂, limits of detection were 0.025, 0.05, 0.05, and 0.075 ng/g and limits of quantification were 0.083, 0.16, 0.16, and 0.23 ng/g, respectively. Accuracy was examined by the determination of the relative recovery of the aflatoxins. The relative recovery of aflatoxins B₁, B₂, G₁, and G₂ were quite satisfactory (between 64.38 and 122.21% for food samples). Relative standard deviations for within laboratory repeatability (n = 6) were in the range from 1.3 to 3.2. The application of this sorbent for the separation and concentration of the mentioned aflatoxins from food samples was examined.     

Fast determination of 14 mycotoxins in chestnut by dispersive solid‐phase extraction coupled with ultra high performance liquid chromatography‐tandem mass spectrometry

A dispersive solid‐phase extraction coupled with ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of T‐2 toxin, penicillic acid, fumonisins B₁, B₂, and B₃, aflatoxins B₁, B₂, G₁, and G₂, ochratoxin A, deoxynivalenol, 3‐acetyldeoxynivalenol, 15‐acetyldeoxynivalenol, and zearalenone in chestnut samples. The method was used to analyze 136 samples obtained from Shandong province in China. The mycotoxins were extracted using a dispersive solid‐phase extraction method and cleaned using an improved quick, easy, cheap, effective, rugged, and safe approach. The mycotoxins were then detected using a triple‐quadrupole mass spectrometer. The limits of detection and quantification ranged from 0.02 to 1 and 0.1 to 2 μg/kg, respectively. The recovery rates ranged from 74.2 to 109.5%, with relative standard deviations below 15%. A total of 71 samples were contaminated with seven mycotoxins at concentrations ranging from 1.2 to 105.5 μg/kg, with a number of samples exceeding the maximum limits set in the European regulations for mycotoxins in unprocessed chestnuts.     

Simultaneous determination of aflatoxins B2 and G2 in peanuts using spectrofluorescence coupled with parallel factor analysis

In the present study a method for the simultaneous determination of aflatoxins B₂ and G₂ in peanuts has been developed. The method uses second order standard addition method and excitation–emission fluorescence data together with parallel factor analysis (PARAFAC). The aflatoxin analysis was based on extraction with methanol–water and carried out using immunoaffinity clean-up. The results of PARAFAC on a set of spiked and naturally contaminated peanuts indicated that the two aflatoxins could be successfully determined. The method was validated and analytical figures of merit were obtained for both analytes. The limits of detection (LOD) were 0.05 and 0.04 μg kg⁻¹ for aflatoxins B₂ and G₂, respectively. The limits of quantification (LOQ) were 0.16 and 0.12 μg kg⁻¹ for aflatoxins B₂ and G₂, respectively. Coupling of spectrofluorimetry with PARAFAC can be considered as an alternative method for quantification of aflatoxins in the presence of unknown interferences obtained through analysis of highly complex matrix of peanuts samples at a reduced cost per analysis.     

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.     

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.

Development of a multi‐mycotoxin liquid chromatography/tandem mass spectrometry method for sweet pepper analysis

A multi‐mycotoxin method was developed for the simultaneous determination of trichothecenes (nivalenol, deoxynivalenol, 3‐acetyldeoxynivalenol, 15‐acetyldeoxynivalenol, neosolaniol, fusarenon‐X, diacetoxyscirpenol, HT‐2 toxin, T‐2 toxin), aflatoxins (aflatoxin‐B₁, aflatoxin‐B₂, aflatoxin‐G₁ and aflatoxin‐G₂), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin‐B₁, fumonisin‐B₂ and fumonisin‐B₃), ochratoxin A, zearalenone, beauvericin and sterigmatocystin in sweet pepper. Sweet pepper was extracted with ethyl acetate/formic acid (99:1, v/v). After splitting up the extract, two‐thirds of the extract was cleaned up using an aminopropyl column followed by an octadecyl column. The remaining part was cleaned up using a strong anion‐exchange column. After recombination of both cleaned parts of the sample extract, the combined solvents were evaporated and the residue was dissolved in mobile phase; 20 µL was injected into the chromatographic system, so only one run was used to separate and detect the mycotoxins in positive electrospray ionization using selected reaction monitoring. The samples were analyzed with a Micromass Quattro Micro triple quadrupole mass spectrometer (Waters, Milford, MA, USA). The mobile phase consisted of variable mixtures of water and methanol, 1% acetic acid and 5 mM ammonium acetate. The limits of detection of the multi‐mycotoxin method varied from 0.32 µg.kg^?1 to 42.48 µg.kg^?1. The multi‐mycotoxin liquid chromatography/tandem mass spectrometry (LC/MS/MS) method fulfilled the method performance criteria required by the Commission Regulation (EC) No 401/2006. Sweet peppers inoculated by Fusarium species were analyzed using the developed method. Beauvericin (9–484 µg.kg^?1) and fumonisins (fumonisin‐B₁ up to 4330 µg.kg^?1, fumonisin‐B₂ up to 4900 µg.kg^?1, and fumonisin‐B₃ up to 299 µg.kg^?1) were detected. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of Aflatoxins in Plant-based Milk and Dairy Products by Dispersive Liquid–Liquid Microextraction and High-performance Liquid Chromatography with Fluorescence Detection

The consumption of plant-based milk has increased due to their nutritional attributes. However, these products may contain aflatoxins if contaminated raw materials were used, although little concern is present in international regulation regarding this topic. In this work, dispersive liquid–liquid microextraction (DLLME) was used for the determination of the most important aflatoxins (B₁, B₂, G₁, and G₂) in oat, rice, coconut, almond, and birdseed plant-based milk and milk-based products enriched with oats, almonds, and walnuts using high-performance liquid chromatography (HPLC) with photochemical derivatization and fluorescence detection. Calibrations in matrix were performed for all of the samples, obtaining satisfactory linearity, with correlation coefficients exceeding 0.994 for all of the aflatoxins. The precision in terms of repeatability and intermediate precision, expressed as the relative standard deviation, was lower than 9.7%, and recoveries ranged between 82 and 104%, fulfilling current legislation for the determination of aflatoxins. In addition, the limits of quantification were 0.5?µg?L^?1 for the aflatoxins, allowing the determination of these compounds below the maximum levels established by European Commission in these commodities. Finally, 23 commercial products were analyzed to characterize the presence of these toxins.     

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.     

Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure

Monoclonal antibodies (McAbs) cross-reactive with four major aflatoxins were achieved using a modified two-step screening procedure. The first step was twice modified indirect enzyme-linked immunosorbent assay (ELISA) and resulted in positive hybridomas and hapten-specific antibodies. The modified indirect competitive ELISA (ciELISA) was the second step, in which the competition incubation time was decreased to 30 min, aflatoxin B₁, B₂, G₁ and G₂ were all used as competitors, the concentrations of four aflatoxins were gradiently decreased in each screening. 2-3 subclonings were performed after every modified fusion and resulted in eight hybridomas that secreted antibodies with good cross-reactivity and high affinity to four aflatoxins. Five McAbs were chosen for further analysis. Of the five, two antibodies had similar reaction efficiency with aflatoxin B₁, B₂ and G₁ but showed a weak cross-reaction to G₂. Another two had almost identical reaction capability with four aflatoxins. One clone 1C11 exhibited the highest sensitivity for all four aflatoxins. The concentrations of aflatoxin B₁, B₂, G₁ and G₂ at 50% inhibition for 1C11 were 1.2, 1.3, 2.2 and 18.0 pg mL⁻¹ respectively. This is the most sensitive for all four major aflatoxins described so far. The results indicated that the modified two-step screening procedure had superiority and these antibodies could be used for simultaneous analysis of total aflatoxins.     

Ionic‐liquid‐based dispersive liquid–liquid microextraction combined with magnetic solid‐phase extraction for the determination of aflatoxins B1, B2, G1, and G2 in animal feeds by high‐performance liquid chromatography with fluorescence detection

A novel two‐step extraction technique combining ionic‐liquid‐based dispersive liquid–liquid microextraction with magnetic solid‐phase extraction was developed for the preconcentration and separation of aflatoxins in animal feedstuffs before high‐performance liquid chromatography coupled with fluorescence detection. In this work, ionic liquid 1‐octyl‐3‐methylimidazolium hexafluorophosphate was used as the extractant in dispersive liquid–liquid microextraction, and hydrophobic pelargonic acid modified Fe₃O₄ magnetic nanoparticles as an efficient adsorbent were applied to retrieve the aflatoxins‐containing ionic liquid. Notably, the target of magnetic nanoparticles was the ionic liquid rather than the aflatoxins. Because of the rapid mass transfer associated with the dispersive liquid–liquid microextraction and magnetic solid phase steps, fast extraction could be achieved. The main parameters affecting the extraction recoveries of aflatoxins were investigated and optimized. Under the optimum conditions, vortexing at 2500 rpm for 1 min in the dispersive liquid–liquid microextraction and magnetic solid‐phase extraction and then desorption by sonication for 2 min with acetonitrile as eluent. The recoveries were 90.3–103.7% with relative standard deviations of 3.2–6.4%. Good linearity was observed with correlation coefficients ranged from 0.9986 to 0.9995. The detection limits were 0.632, 0.087, 0.422 and 0.146 ng/mL for aflatoxins B₁, B2, G1, and G2, respectively. The results were also compared with the pretreatment method carried out by conventional immunoaffinity columns.     

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.     

Development of an economic ultrafast liquid chromatography with tandem mass spectrometry method for trace analysis of multiclass mycotoxins in Polygonum multiflorum

Rapid, economic, and highly effective determination of multiple mycotoxins in complex matrices has given huge challenges for the analytical method. In this study, an economic analytical strategy based on sensitive and rapid ultrafast liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometry technique was developed for the determination of seven mycotoxins of different chemical classes (aflatoxin B₁, B₂, G₁, and G₂, ochratoxin A, T‐2 toxin, and HT‐2 toxin) in Polygonum multiflorum. Target mycotoxins were completely extracted using a modified quick, easy, cheap effective, rugged, and safe method without additional clean‐up steps. The types of extraction solvents and adsorbents for the extraction procedure were optimized to achieve high recoveries and reduce coextractives in the final extracts. Due to significant matrix effects for all analytes (≤68.9% and ≥110.0%), matrix‐matched calibration curves were introduced for reliable quantification, exploring excellent linearity for the seven mycotoxins with coefficients of determination >0.9992. The method allowed high sensitivity with limit of detection in the range of 0.031–2.5 μg/kg and limit of quantitation in the range of 0.078–6.25 μg/kg, as well as satisfactory precision with relative standard deviations lower than 8%. Recovery rates were between 74.3 and 119.8% with relative standard deviations below 7.43%. The proposed method was successfully applied for 24 batches of P. multiflorum samples, and six samples were found to be positive with aflatoxin B₁, B₂, G₁, or ochratoxin A. The method with significant advantages, including minimum analytical time, low time and solvent consumption, and high sensitivity, would be a preferred candidate for economic analysis of multiclass mycotoxins in complex matrices.     

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.

Validation of analytical methods for determining mycotoxins in foodstuffs

The European Union (EU) has established demanding regulatory limits for controlling aflatoxins B₁, B₂, G₁ and G₂, in cereals, nuts, nut products and dried fruit, aflatoxin M₁ in milk, and ochratoxin A in cereals. These limits are likely to be extended in the future to additional commodities and other mycotoxins. For enforcement purposes and in particular for resolving any disputes between parties, it is essential that validated methods are available, with performance characteristics that meet certain minimum criteria. As such methods were not available and had not previously been validated either for matrices of interest in Europe or at the low European limits compared to the USA, the EU funded a method-validation project to fulfil this requirement. Immunoaffinity column clean-up methods with HPLC determination were established for aflatoxins B₁, B₂, G₁ and G₂ in peanut butter, pistachios, fig paste and paprika, aflatoxin B₁ in baby food, aflatoxin M₁ in liquid milk, and ochratoxin A in roasted coffee and baby food. For patulin in apple juice and apple puree, solvent extraction and solid-phase clean-up HPLC methods were developed. To undertake collaborative studies, particular care was taken in preparation of naturally-contaminated test materials containing the toxins at levels close to regulatory limits and in demonstrating the homogeneity of batches of material. To ensure that participants in the validation exercise could follow the procedures to be tested, videos of the methods were prepared showing, in particular, any critical steps. Prior to undertaking the method validation, participants were invited to collaborative study workshops to ensure that they fully understood the methods and their role in the study. This care in planning and executing the collaborative studies led to impressive performance characteristics and adoption of six procedures by AOAC International as First Action Methods and seven methods by CEN as European standards. The valuable lessons learned in undertaking these validation exercises are now being put to further use in studies aimed at validating methods for mycotoxins in foodstuffs, which are appropriate for developing countries based on TLC as the end determination but use more modern sample clean-up techniques.     

A Guide to Thin-Layer Chromatographic Systems for the Separation of Aflatoxin B1, B2, G1 and G2

Abstract

The separation of aflatoxin B₁, B₂, G₁ and G₂ was compared on six commercial silica gel plates in twelve solvent systems. Two of the solvent systems, chloroform: acetone: ammonium hydroxide (90: 10: 0.25) and chloroform: acetone: hexane (85: 15: 20) resolved the four aflatoxins on all the tested plates. The solvent modifier played an important role in the resolution of these compounds. The effect of the hardness of the plate is also discussed.