Use of liquid chromatography–high‐resolution mass spectrometry for isolation and characterization of hydrolyzed fumonisins and relevant analysis in maize‐based products

The synthesis of partially hydrolyzed fumonisins (PHFB₁ and PHFB₂) and hydrolyzed fumonisins (HFB₁ and HFB₂) by chemical hydrolysis of pure fumonisins (FB₁ and FB₂) is reported together with the isolation and characterization by liquid chromatography–high‐resolution mass spectrometry (LC–HRMS). Two structural isomers of partially hydrolyzed forms of FB₁ and FB₂ were identified, namely PHFB_1a and PHFB_1b and PHFB_2a and PHFB_2b. Reaction yields were 21% for PHFB₁ (sum of the two isomers), 52% for HFB₁, 31% for PHFB₂ (sum of the two isomers) and 30% for HFB₂. Purity of each isolated compound was >98%. An LC–HRMS method for the simultaneous determination of fumonisins and their partially and totally hydrolyzed derivatives was applied to 24 naturally contaminated samples of maize and maize‐based products. The majority of samples (18 out of 24) were contaminated with fumonisins B₁ and B₂. Fumonisins co‐occurred with both partially hydrolyzed and hydrolyzed fumonisins in four nixtamalized samples (three masa flours and one tortilla chips). Co‐occurrence of fumonisins with partially hydrolyzed fumonisins was also recorded in one sample of maize kernels and four samples of maize‐based products (i.e. maize meal, cous‐cous, corn‐cakes and cornflakes). Mycotoxins levels ranged from 60 to 5700 µg/kg for fumonisins (sum of FB₁ and FB₂), from 10 to 210 µg/kg for partially hydrolyzed fumonisins (sum of PHFB₁ and PHFB₂) and from 30 to 200 µg/kg for hydrolyzed fumonisins (sum of HFB₁ and HFB₂). This is the first report of the isolation of PHFB₂ and the co‐occurrence of FB_1, FB₂, PHFB₁, PHFB₂, HFB₁ and HFB₂ in maize products. Considering the growing use of nixtamalized and maize‐based products, the monitoring of fumonisins and their partially and totally hydrolyzed forms in these products may represent an important contributing factor in evaluating the relevant human risk exposure. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Difficulties in fumonisin determination: the issue of hidden fumonisins

In this paper, the results obtained by five independent methods for the quantification of fumonisins B₁, B₂, and B₃ in raw maize are reported. Five naturally contaminated maize samples and a reference material were analyzed in three different laboratories. Although each method was validated and common calibrants were used, a poor agreement about fumonisin contamination levels was obtained. In order to investigate the interactions among analyte and matrix leading to this lack of consistency, the occurrence of fumonisin derivatives was checked. Significant amounts of hidden fumonisins were detected for all the considered samples. Furthermore, the application of an in vitro digestion protocol to raw maize allowed for a higher recovery of native fumonisins, suggesting that the interaction occurring among analytes and matrix macromolecules is associative rather than covalent. Depending on the analytical method as well as the maize sample, only 37–68% of the total fumonisin concentrations were found to be extractable from the samples. These results are particularly impressive and significant in the case of the certified reference material, underlying the actual difficulties in ascertaining the trueness of a method for fumonisin determination, opening thus an important issue for risk assessment.     

Determination of fumonisins B1 and B2 in Portuguese maize and maize-based samples by HPLC with fluorescence detection

Fumonisins B₁ (FB₁) and fumonisin B₂ (FB₂) are the main members of a family of mycotoxins produced by Fusarium verticillioides, Fusarium proliferatum, and other fungi species of the section Liseola. The present work shows the results of comparative studies using two different procedures for the analysis of fumonisins in maize and maize-based samples. The studied analytical methods involve extraction with methanol/water, dilution with PBS, and clean-up through immunoaffinity columns. Two reagents (o-phthaldialdehyde and naphthalene-2,3-dicarboxaldehyde) were studied for formation of fluorescent derivatives. The separation and identification were carried out by high-performance liquid chromatography with fluorescence detection. The optimized method for analysis of fumonisins in maize involved extraction with methanol/water (80:20), clean-up with an immunoaffinity column, and derivatization with naphthalene-2,3-dicarboxaldehyde (NDA). The limit of detection was 20 μg kg⁻¹ for FB₁ and 15 μg kg⁻¹ for FB₂. Recoveries of FB₁ and FB₂ ranged from 79% to 99.6% for maize fortified at 150 μg kg⁻¹ and 200 μg kg⁻¹, respectively, with within-day RSDs of 3.0 and 2.7%. The proposed method was applied to 31 samples, and the presence of fumonisins was found in 14 samples at concentrations ranging from 113 to 2,026 μg kg⁻¹. The estimated daily intake of fumonisins was 0.14 μg kg⁻¹ body weight per day.     

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.     

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.     

Incidence of Fusarium Mycotoxins in Wheat and Maize from Albania

In this study, ten Fusarium toxins were analysed in wheat and maize commodities from Albania. In total, 71 samples of wheat and 45 samples of maize were collected from different producing regions. The analytical procedure consisted of a simple one-step sample extraction followed by the determination of toxins using liquid chromatography coupled with tandem mass spectrometry. Fusarium toxins were found in 23% of the analysed wheat samples and in 78% of maize samples. In maize samples, most often fumonisins B₁ (FB1) and B₂ (FB2) were found. They were present in 76% of samples. They were detected in all positive samples except in one with concentrations ranging from 59.9 to 16,970 μg/kg. The sum of FB1 and FB2 exceeded the EU maximum permitted level (4000 μg/kg) in 31% of maize samples. In wheat samples, the only detected Fusarium mycotoxin was deoxynivalenol (DON), present in 23% of samples. In one sample with the concentration of 1916 μg/kg, the EU maximum permitted level (1250 μg/kg) was exceeded. This is the first report on the presence of Fusarium toxins in wheat and maize grains cultivated in Albania.     

Analysis of 16 phthalic acid esters in food simulants from plastic food contact materials by LC‐ESI‐MS/MS

An RP LC‐ESI‐MS/MS method for the determination of the migration of 16 primary phthalic acid esters from plastic samples has been developed using distilled water, 3% acetic acid, 10% alcohol, and olive oil as food simulants. Detection limits were 1.6–18.5 μg/kg in distilled water, 1.4–17.3 μg/kg in 3% acetic acid, 1.4–19.2 μg/kg in 10% alcohol, and 31.9–390.8 μg/kg in olive oil. The RSDs were in the range of 0.07–11.28%. The real plastic products inspection showed that only few analyzed samples were phthalates contaminated. Bis‐2‐ethylhexyl ester and dibutyl phthalate were the common items migrated from the plastic products into food and feeds, but the migration concentrations were far below the limits set by European Union (1.5 mg/kg for bis‐2‐ethylhexyl ester and 0.3 mg/kg for dibutyl phthalate).     

Rapid screening of amitraz and its metabolite residues in honey using a quick,easy, cheap,effective, rugged,and safe extraction method coupled with UHPLC and Q Exactive

A method for determining amitraz and 2,4‐dimethylaniline in honey was established by using ultra‐high‐performance liquid chromatoghaphy and Q Exactive after applying quick, easy, cheap, effective, rugged, and safe extracting process. A suitable extraction method was designed to extract the amitraz and 2,4‐dimethylaniline after a suitable amount of honey samples was dissolved. A Thermo Syncronis C₁₈ column (100 × 2.1 mm, 1.7 μm) was used for chromatographic separation of the samples. Then the two compounds were quantitatively analyzed via a program of Q Exactive. The linearity of amitraz and 2,4‐dimethylaniline was good in the concentration range of 0.5–100 μg/L, and the correlation coefficient R² was >0.99. The average recovery and relative standard deviation of each component were 81.3–90.0% and 5.1–7.2%. The 24‐ and 48‐h test results showed that the sample needed to be tested within 24 h. The limit of detection was 0.1 μg/kg for amitraz and 2,4‐dimethylaniline, whereas for both the limit of quantitation was 0.3 μg/kg.     

Simultaneous Spectrophotometric Determination of Group B Vitamins Using Parallel Factor Analysis: PARAFAC

In this work, a simple and rapid analytical procedure was applied for simultaneous determination of folic acid (vitamin B₀), thiamin (vitamin B₁), riboflavin (vitamin B₂) and pyridoxal (vitamin B₆) based on the absorbance data in the pH range 2.0‐12.0 at 25 °C using parallel factor analysis (PARAFAC). The effect of the pH as the most important factor on the sensitivity of the determination was studied. The spectral data were recorded in 400‐650 nm intervals and a 2‐12 pH range for all four vitamins. The calibration set was constructed in the concentration ranges of 4‐22, 1‐20, 6‐26, and 4‐20 μg mL^?;1 for B₆,B₂,B₁ and B₀, respectively. The root mean squares errors of prediction for the prediction set, (RMSEP), are 0.65, 0.63, 1.13 and 0.34 for B₀,B₁,B₂ and B₆, respectively. The recovery percent for the validation set are in the range of 90.6 to 107.0%. The effect of the experimental conditions and diverse species were discussed. The optimum values of these factors were searched according to the relative standard deviation of the prediction set of mixtures solutions.     

Fumonisins determination in urine by LC-MS-MS

The present work shows the optimization and validation results of an analytical methodology based on imunoaffinity clean-up (IAC) followed by liquid chromatography with tandem mass detection (LC-MS-MS) for the analysis of the mycotoxins fumonisin B₁ (FB₁) and B₂ (FB₂) in human urine samples, in order to evaluate fumonisins exposure in two Portuguese populations. As far as we know, imunoaffinity clean-up procedure was used, for the first time, in the analysis of fumonisins (FBs) in urine. Using this analytical methodology, the limit of quantification achieved was 10 ng mL⁻¹ for FB₁ and for FB₂. Recoveries were higher than 73.4% for fortification levels between 10 and 100 ng mL⁻¹ and intra-day and inter-day repeatibility were lower than 8.6%. The natural occurrence of FB₁ and FB₂ in 68 human urine samples obtained from the central zone of Portugal was studied. None of the studied samples presented detectable levels of FB₁ and FB₂.     

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.     

DNA Electrochemical Aptasensor for Detecting Fumonisins B1 Based on Graphene and Thionine Nanocomposite

In this paper, a novel aptasensor was designed by with the dual amplification of Au nanoparticles (AuNPs) and graphene/thionine nanocomposites (GS‐TH) for sensitive determination of fumonisins B₁ (FB₁). AuNPs is modified at the electrode surface to increase the electrical conductivity and fabricate specific recognition interface for FB₁ through the hybridization of capture DNA and its aptamer. Large number of TH molecules were loaded at the surface of graphene sheet to served as electrochemical probe and increase its electrochemical signal due to the excellent conductivity and large surface area of graphene sheet. This type of nanocomposites is then assembled to the single strand section of FB₁ aptamer at electrode surface by π–π stacking interactions between them, leading to an enhanced electrochemical signal. After the specific combination between FB₁ aptamer and its target (FB₁) in solution, GS–TH was released from electrode surface, resulting in a decreased electrochemical signal. The result demonstrated that the decreased currents were proportional to the FB₁ concentration in the range of 1–10⁶ pg/mL with a detection limit of 1 pg/mL. Besides, the developed aptasensor was also applied successfully for the determination of FB₁ in feed samples. The result shows this aptasensor has a higher sensitivity and selectivity.     

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 major type‐B trichothecenes and the de‐epoxy metabolite of deoxynivalenol in chicken tissues by HPLC–MS/MS

In this study, a specific and sensitive LC–MS/MS method for the simultaneous analysis of type‐B trichothecenes (deoxynivalenol, 3‐acetyldeoxynivalenol, and 15‐acetyldeoxynivalenol) and the de‐epoxy metabolite of deoxynivalenol (de‐epoxy‐deoxynivalenol) in chicken muscle, liver, kidney, and fat tissues was developed and validated. The method involved an extraction step using ethyl acetate, followed by the evaporation of the supernatant, which was further purified by an Oasis HLB SPE cartridge (Waters, Milford, MA, USA). Chromatographic separation was performed on a C₁₈ column by detection with MS in multiple‐reaction monitoring mode and using a gradient elution program with 0.1% formic acid in water and methanol. The correlation coefficients (r) for each calibration curve were >0.99 within the experimental concentration range. The extraction recoveries ranged from 73.7 to 106.4%, with intraday and interday RSD < 11.6% at three levels of concentrations of 2, 10, and 100 μg/kg. The decision limits and the detection capabilities of the analytes in the chicken tissues ranged from 0.16 to 0.92 and 0.68 to 2.07 μg/kg, respectively. The results demonstrated the applicability of this sensitive procedure to the determination of trichothecenes in chicken tissue samples.     

Simultaneous determination of pyrametostrobin and its two metabolites in cucumber and soil using a quick,easy, cheap,effective, rugged,and safe method with HPLC–MS/MS

An easy, effective and sensitive analytical method for the simultaneous determination of a novel fungicide pyrametostrobin and its two metabolites pyrametostrobin‐M1 and pyrametostrobin‐M2 in cucumber and soil was developed using a quick, easy, cheap, effective, rugged, and safe method with high‐performance liquid chromatography and tandem mass spectrometry. The extraction solvent was acetonitrile, and cleanup sorbents were primary secondary amine and graphitized carbon black for cucumber samples and primary secondary amine for soil samples. The three target compounds were successfully separated between 3.2 and 3.9 min using a Waters CORTECS™ C₁₈ column connected to an electrospray ionization source. All the matrix‐matched samples at three fortified levels (10, 100 and 1000  μg/kg) provided satisfactory recoveries in the range of 78.8–93.8% with relative standard deviations below 6.9%. The limits of quantitation for the three compounds were below 0.183 μg/kg. The proposed method was validated by analyzing real samples.     

Response surface methodology approach for the preparation of a molecularly imprinted polymer for solid‐phase extraction of fenoxycarb pesticide in mussels

The aim of this work was to develop an efficient method for the selective extraction and analysis of fenoxycarb, a carbamate pesticide, in mussel samples using a molecularly imprinted solid‐phase extraction device. The optimization of molecularly imprinted polymer synthesis was performed using the experimental design under the response surface methodology approach. A fast rebinding study and Freundlich isotherm adsorption were carried out to calculate binding capacity B, site number n, and affinity constant K_f. The optimum molecularly imprinted polymer was successfully used as sorbent of a solid‐phase extraction cartridge for the determination of fenoxycarb in real mussel samples. The range of linearity was 0.3–30 mg/L with a correlation coefficient of 0.991. The limit of detection was 0.247 mg/kg. The recovery of fenoxycarb extracted from mussel samples of Mediterranean sea was 97% (n = 3) with relative standard deviation between 6 and 7% proving the reliability of the developed method.     

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.     

A Highly Sensitive Electrochemical Immunosensor for Fumonisin B1 Detection in Corn Using Single‐Walled Carbon Nanotubes/Chitosan

A sensitive electrochemical immunosensor was developed for detecting fumonisin B₁ (FB₁) in corn using the single‐walled carbon nanotubes/chitosan. The detection mechanism of immunosensor was based on an indirect competitive binding to a fixed amount of anti‐FB₁ between free FB₁ and FB₁‐bovine serum albumin, which was conjugated on covalently functionalized nanotubes/chitosan laid on the glass carbon electrode. The anti‐rabbit immunoglobulin G secondary antibody labeled with alkaline phosphatase was then bound to the electrode surface through reactisubstrate α‐naphthyl phosphate, which produced electrochemical signal. Under optimized conditions, this method could detect FB₁ from 0.01 to 1000 ng mL^?1 with a detection limit of 2 pg mL^?1. This is well below the detection limit required from European Union legislation, 2–4 mg L^?1. Moreover, good recoveries were obtained for the detection of spiked corn samples and actual corn samples. As the method has good sensitivity and recovery for detecting FB₁, it is a practical detection method.     

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.