Simultaneous Determination Of Aflatoxin B1 Research Articles

Simultaneous determination of aflatoxin B1, aflatoxin B2, mycophenolic acid and sterigmatocystin in grape pomace by UHPLC-MS/MS

A reliable ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of aflatoxin B1, aflatoxin B2, mycophenolic acid and sterigmatocystin in grape pomace. The samples were extracted by acetonitrile aqueous solution and further purified using a solid-phase extraction-based homemade clean-up cartridge. Next, the analytes were separated on a reversed-phase C18 column with a mobile phase consisting of water and acetonitrile. The separated compounds were detected with a tandem quadrupole mass spectrometer operating in positive electro-spray ionisation mode using multiple reaction monitoring. The established method was extensively validated by determining linearity (R2≯0.999), recovery (97.5-102.8%) and precision (relative standard deviation ≤7.0%). This method was then used for the simultaneous determination of the four mycotoxins in grape pomace samples.

DETERMINATION OF AFLATOXIN B1 AND B2 IN PEANUT AND PEANUT OIL USING CLOUD POINT EXTRACTION FOLLOWED BY ULTRA-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

A new approach for ultra-high-performance liquid chromatography with fluorescence detection (UHPLC-FLD) determination of aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) in peanut and peanut oil by cloud point extraction (CPE) has been developed. The nonionic surfactant Triton X-114 was chosen to extract the target aflatoxin. The different parameters affecting the extraction of target analytes such as pH, concentration of surfactant, and temperature were investigated and optimized. Linear calibration curves were obtained in the range of 1.0–1000.0 ng/mL under optimum conditions. Recoveries ranged of 90.17–96.25% and the relative standard deviations (RSD) were 1.13–4.12% (n = 5). All correlation coefficients of the calibration curves were higher than 0.999. The intra-day precision for 1.13–4.12% and inter-day precision for 1.30–5.12%, and the limits of detections (LODs) were 0.40 ng/mL and 0.20 ng/mL for AFB1 and AFB2, respectively. And, the concentration factor was 20. The method was applied to the simultaneous determination of AFB1 and AFB2 in peanut and peanut oil.

Simultaneous determination of aflatoxin B1 and ochratoxin A in licorice roots and fritillary bulbs by solid-phase extraction coupled with high-performance liquid chromatography–tandem mass spectrometry

An effective method was developed for screening licorice roots and fritillary bulbs for contamination by aflatoxin B1 and ochratoxin A using high-performance liquid chromatography connected to tandem mass spectrometry (HPLC–MS/MS). The samples were pre-concentrated and purified using solid-phase extraction, which provided satisfactory results. The separation was performed on a Waters XbridgeTM C18 column with a linear gradient of acetonitrile - water containing 5mM ammonium acetate. The MS spectrum was acquired in positive mode with both single quadrupole (Q1) and multiple reaction monitoring (MRM). The optimised method offered a good linear correlation (r2>0.9967), excellent precision (RSD<2.83%) and acceptable recovery (from 92.78 to 105.37%). The limits of detection (LOD) and the limits of quantification (LOQ) were less than 0.024μg/kg and 0.095μg/kg, respectively. The validated method was successfully applied to the rapid screening for AFB1 and OTA in licorice roots and fritillary bulbs.

Simultaneous Determination of Aflatoxin B1 and Aflatoxin M1 in Food Matrices by Enzyme-Linked Immunosorbent Assay

Aflatoxins are a class of highly toxic chemical contaminants occurring in food. Consumption of aflatoxin-contaminated food can lead to harmful effects on human health. A rapid and reliable analysis of aflatoxins in food is crucial. In this study, we generated a broad–specific monoclonal antibody (MAb) 3 C10 against aflatoxin B1 (AFB1). The MAb 3 C10 binds specifically to AFB1 and AFM1 and has a IC50 of 0.13 μg L−1 for AFB1 and 0.16 μg L−1 for AFM1. Furthermore, the MAb showed high cross-reactivity to AFB2, AFG1, and AFG2. To enable simultaneous AFB1 and AFM1 detection in different food matrices, an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on MAb 3 C10 has been developed and optimized. In addition, the extraction methods of different food matrices (peanut, corn, soybean, wheat flour, rice, soy sauce, vinegar, wine, raw milk, pure milk, skimmed milk, and yogurt) were established. The average recovery ranged from 73 to 121 %, with relative standard deviation values less than 15 %. The limit of detection was 0.52 + 0.36 μg kg−1 (mean + 3SD) for AFB1 in eight agricultural products and 0.031 + 0.015 μg kg−1 (mean + 3SD) for AFM1 in four dairy products. The sensitivity of icELISA was below the limit set by the European Commission for aflatoxin detection in different food matrices and similar to LC–MS/MS method. We demonstrate a rapid, simple, and reliable method for simultaneous screening of AFB1 and AFM1 in different food matrices.

Development of a sensitive and robust liquid chromatography coupled with tandem mass spectrometry and a pressurized liquid extraction for the determination of aflatoxins and ochratoxin A in animal derived foods

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) with a pressurized liquid extraction (PLE) was developed for the simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2, and ochratoxin A in the muscle, liver, kidney and fat of swine, bovine and sheep, muscle and liver of chicken, muscle and skin of fish, as well as in hen eggs and dairy milk. Samples were extracted with PLE and cleaned-up with solid phase extraction (SPE) on HLB cartridges. The optimum extraction conditions were obtained as a 11ml ASE cell, acetonitrile/hexane as the extraction solvent, 1500psi, 100°C, a 5min static time and a 60% flush volume. A cheaper and widely used SPE column (Oasis HLB) was applied during clean up. The detection and quantification of the 7 mycotoxins were performed by a reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS). The limits of detection defined as CCα varied from 0.07μg/kg to 0.59μg/kg. The recoveries of spiked samples from 0.25μg/kg to 1μg/kg ranged from 68.3% to 105.7% with the relative standard deviations of less than 17.6%. Performances of the whole analytical procedure met the criteria established by the European Commission for mass spectrometric detection.

Development and validation of an accurate and rapid LC-ESI-MS/MS method for the simultaneous quantification of aflatoxin B1, B2, G1 and G2 in lotus seeds

An accurate and rapid LC-ESI-MS/MS analytical method was developed and validated for the simultaneous determination of aflatoxin B1, B2, G1 and G2 in lotus seeds. The samples were firstly extracted with methanol-water solution (80:20, v/v), and then cleaned up by immunoaffinity columns. The mass spectrometer was operated in the positive ionization electrospray (ESI+) mode using multiple reaction monitoring (MRM) for analysis of four aflatoxins. The transitions of m/z 313 → 285 (AFB1, CE 33 eV), m/z 315 → 259 (AFB2, CE 37 eV), m/z 329 → 243 (AFG1, CE 37 eV) and m/z 331 → 257 (AFG2, CE 37 eV) were used to quantify these four aflatoxins, respectively. The limits of detection (LODs) of aflatoxin B1, B2, G1 and G2 were 0.007, 0.005, 0.003 and 0.005 μg kg−1 based on a signal-to-noise ratio of 3:1, respectively. The limits of quantification (LOQs) of aflatoxin B1, B2, G1 and G2 were 0.02, 0.015, 0.01 and 0.015 μg kg−1 based on a signal-to-noise ratio of 10:1, respectively. Recoveries for samples of spiked lotus seeds were all above 66% with relative standard deviation all below 15% for all compounds. Nineteen out of twenty batches of lotus seeds collected from different drug stores or markets in China were found to be contaminated with aflatoxins at different levels ranging from 0.02 to 688.4 μg kg−1.

Simultaneous determination of aflatoxin B1, B2, G1, G2 and ochratoxin A in Glycyrrhiza uralensis by HPLC-FLD after immunoaffinity column with online post-column photochemical derivatization

To develop a method for the simultaneous determination of aflatoxin B1, B2, G1, G2 and ochratoxin A in Glycyrrhiza uralensis by HPLC-FLD after immunoaffinity column with online post-column photochemical derivatization. Sample was extracted with MeOH: H2O (80:20) and cleaned up by immunoaffinity column. The toxins were separated by reversed-phase HPLC and the mobile phase was consisted of methanol and 0.5% acetic acid solution with gradient elution. The determination was carried out by fluorescence detector after photochemical derivatization. The detection limits of aflatoxin G2, G1, B2, B1 and ochratoxin A were 0.02, 0.06, 0.015, 0.03 and 0.25 microg x kg(-1), respectively. The recoveries of analytes were from 76.0% to 103% and the relative standard deviations (RSDs) were below 13%. The method is a simple, accurate and can be used to determine the contents of aflatoxin B1, B2, G1, G2 and ochratoxin A in G. uralensis simultaneously.

Optimization and validation of a HPLC method for simultaneous determination of aflatoxin B1, B2, G1, G2, ochratoxin A and zearalenone using an experimental design

A reversed-phase HPLC optimization strategy is presented for investigating the separation and retention behavior of aflatoxin B1, B2, G1, G2, ochratoxin A and zearalenone, simultaneously. A fractional factorial design (FFD) was used to screen the significance effect of seven independent variables on chromatographic responses. The independent variables used were: (X1) column oven temperature (20–40°C), (X2) flow rate (0.8–1.2 ml/min), (X3) acid concentration in aqueous phase (0–2%), (X4) organic solvent percentage at the beginning (40–50%), and (X5) at the end (50–60%) of the gradient mobile phase, as well as (X6) ratio of methanol/acetonitrile at the beginning (1–4) and (X7) at the end (0–1) of gradient mobile phase. Responses of chromatographic analysis were resolution of mycotoxin peaks and HPLC run time. A central composite design (CCD) using response surface methodology (RSM) was then carried out for optimization of the most significant factors by multiple regression models for response variables. The proposed optimal method using 40°C oven temperature, 1 ml/min flow rate, 0.1% acetic acid concentration in aqueous phase, 41% organic phase (beginning), 60% organic phase (end), 1.92 ratio of methanol to acetonitrile (beginning) and 0.2 ratio (end) for X1–X7, respectively, showed good prediction ability between the experimental data and predictive values throughout the studied parameter space. Finally, the optimized method was validated by measuring the linearity, sensitivity, accuracy and precision parameters, and has been applied successfully to the analysis of spiked cereal samples.

An ultra-high-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines (TCMs) was developed. The approach was characterized in details and a special focus was placed on the recovery rates of isolation procedure in different TCM matrices, i.e. rhizomes and roots, seeds, flowers, grasses and leaves. For this purpose, [ 13C 17]-aflatoxinB1 was employed as the internal standard and a reliable solid phase extraction-based clean-up method was developed. The observed recovery rates of the six aflatoxins ranged from 85.6% to 117.6% in different matrices. Then, the established method was successfully applied to the determination of the six aflatoxins in various TCMs. For 30 commercial samples analyzed, 16 were contaminated with aflatoxins. The mean levels (incidence) of aflatoxins B1, B2, G1 and G2 in positive samples were 1.40 (68.8%), 1.27 (50.0%), 0.50 (43.8%) and 0.94 (43.8%) μg kg −1, respectively. Interestingly, aflatoxin M1 was detected in two samples with the maximal content of 0.70 μg kg −1. No sample was contaminated with aflatoxin M2. Meanwhile, a possible association between the contamination levels and the selected herbs was clarified in the present study.

Simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products by ultra-high-performance liquid chromatography–tandem mass spectrometry

A reliable ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products was developed. The sample was extracted by 84% of acetonitrile aqueous solution and the extract was purified by a reliable solid phase extraction-based clean-up method. Then, the analytes were separated on Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm particle size), and eluted with a mobile phase consisting of (A) water containing 0.1% formic acid and (B) acetonitrile/methanol (50/50, v/v). The separated compounds were detected with a Waters Micromass Quattro Ultima Pt tandem quadrupole mass spectrometer operating in positive electro-spray ionization using multiple reaction monitoring mode. The established method was extensively validated by determining the linearity ( R 2 ≥ 0.9990), average recovery (74.7–86.8%) and precision (relative standard deviation ≤ 10.9%). It was shown to be a suitable method for simultaneous determination of the six aflatoxins in peanuts and their derivative products. Finally, a total of 73 samples randomly collected from different areas in Zhejiang province were screened for aflatoxins with the proposed method. The results showed that 31 samples of peanut butter, 14 samples of fresh peanut and 5 samples of musty peanut were contaminated with aflatoxins. Meanwhile, this was the first report on aflatoxins M1 and M2, which were found in unprocessed peanuts and their derivative products.

Comparison and improvement of the existing methods for the determination of aflatoxins in human serum by LC-MS/MS

Aflatoxins are toxic metabolites of some Aspergillus flavus, A. parasiticus and A. nomius strains that occur in many foods and feeds. Aflatoxin B1 is the most abundant and toxic member of the family, and it is also the most potent hepatocarcinogen known. In order to estimate the potential human health risk of aflatoxin B1, it is useful to measure its blood concentration. In this work a rapid liquid chromatography coupled to mass spectrometry (LC-MS/MS) method for the simultaneous determination of aflatoxins B1, B2, G1 and G2 and its metabolites, aflatoxins M1 and M2 in human serum, is proposed. Small serum volumes have been used and the extracts have been obtained without any clean-up procedure. The isolation of the aflatoxins from human serum was achieved using subsequent pH-controlled solvent extractions steps. Recovery rates in the range between 31 and 98% were achieved in the serum, while the limits of detection and quantification for the proposed method were 0.5 ng mL−1 and 1.0 ng mL−1, respectively. The validation of the method has been demonstrated by analysing spiked and naturally contaminated blood serum.

Simultaneous determination of aflatoxin B1 and ochratoxin A and their natural occurrence in Mediterranean virgin olive oil

Olive oil, the most important dietary fat source of the Mediterranean diet, can be contaminated by mycotoxins. An efficient analytical method for the simultaneous determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in olive oil is reported. Thirty commercial samples of virgin olive oil, purchased in olive-press plants and supermarkets in southern Italy and North Africa, were analysed to verify the analytical procedure and monitor mycotoxin contamination. A simple, rapid and economic method was set up and tested for both the extractive step and the clean-up procedures for simultaneous AFB1 and OTA determination in olive oil. Data obtained showed that OTA was detected with high frequency (80%) in samples from both geographical areas (up to 17.0 ng g–1), while AFB1 was found from three of four samples from North Africa (up to 2.4 ng g–1). In addition, ‘not labelled’ oil samples proved to be more contaminated by OTA then ‘labelled’ samples (mean values of 2.47 and 0.66 ng g–1, respectively). These findings indicate that olive oil can be significantly contaminated by mycotoxins and confirm that a scrupulous application of European Regulation 1019/2002 (European Commission 2002), which prohibits the sale of non-labelled olive oil, is strongly recommended. Conventional qualitative parameters such as peroxide number, spectrophotometric evaluation and acid values were not correlated with mycotoxin occurrence.

Immunoaffinity Column Clean-Up and Liquid Chromatography with Post-Column Derivatization for Analysis of Aflatoxins in Traditional Chinese Medicine

An easy, rapid, and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method with fluorescence detection is proposed for simultaneous determination of aflatoxins B1, B2, G1, and G2 in traditional Chinese medicines. Aflatoxins B1 and G1 were determined as their iodine derivatives, prepared by on-line post-column derivatization. Optimum conditions for formation of the derivatives were investigated. The aflatoxins were separated on a C18 column with acetonitrile–methanol–water, 1:1:4 (v/v), as mobile phase at a flow rate of 1.0 mL min−1. Stability of solutions, and linearity, accuracy, precision, and detection limits were examined to validate the method. Samples were extracted with MeOH–H2O, 70:30 (v/v), followed by clean-up of the extracts with immunoaffinity columns. Overall average recoveries from three different traditional Chinese medicines spiked at levels of 2.57, 5.10, and 6.37 µg kg−1 total aflatoxins ranged from 84.49–103.77%. The method was tested on Rhizoma Corydalis, Fujian Massa Medicata Fermentata, Semen Armenicae Amarum, Herba Ephedrae, Semen Platycladi, etc.

Simultaneous Thin Layer Chromatographic Determination of Aflatoxin B1 and Ochratoxin A in Black Olives

A screening method has been developed for simultaneous determination of aflatoxin B1 and ochratoxin A in black olives. The technique includes extraction of both mycotoxins with aqueous methanol, cleanup using lead acetate, defatting with hexane, partitioning in chloroform, and thin layer chromatography. Detection limits are 5-7 micrograms aflatoxin B1 and 20 micrograms ochratoxin A/kg.

Simultaneous detection of aflatoxin b1 and ochratoxin a in olive oil

AbstractA screening method has been developed for simultaneous determination of aflatoxin B1 and ochratoxin A in olive oil. The technique includes extraction of both mycotoxins with aqueous methanol, clean‐up using lead acetate, partitioning in chloroform, and thin layer chromatography. The detection limits achieved are, respectively: 4 μg aflatoxin B1 and 40 μg ochratoxin A/kg. 60 samples of crude farm olive oil were analyzed using this method: three of them contained traces of ochratoxin A while none was contaminated with aflatoxin B1.