Christof Van Poucke

Occurrence of Mycotoxins in Feed as Analyzed by a Multi-Mycotoxin LC-MS/MS Method

Crops used for animal feed can be easily contaminated by fungi during growth, harvest, or storage, resulting in the occurrence of mycotoxins. Because animal feed plays an important role in the food safety chain, the European Commission has set maximum levels for aflatoxin B1 and recommended maximum levels for deoxynivalenol, zearalenone, ochratoxin A, and the sum of fumonisin B1 and B2. A multimycotoxin LC-MS/MS method was developed, validated according to Commission Decision 2002/657/EC and EN ISO 17025 accredited for the simultaneous detection of 23 mycotoxins (aflatoxin-B1, aflatoxin-B2, aflatoxin-G1, aflatoxin-G2, ochratoxin A, deoxynivalenol, zearalenone, fumonisin B1, fumonisin B2, fumonisin B3, T2-toxin, HT2-toxin, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, fusarenon-X, neosolaniol, altenuene, alternariol, alternariol methyl ether, roquefortine-C, and sterigmatocystin) in feed. The decision limits of the multimycotoxin method varied from 0.7 to 60.6 microg/kg. The apparent recovery and the results of the precision study fulfilled the performance criteria as set in Commission Decision 2002/657/EC. The analysis of three different feed matrices (sow feed, wheat, and maize) provided a good basis for the evaluation of the toxin exposure in animal production. In total, 67 samples out of 82 (82%) were contaminated; type B-trichothecenes and fumonisins occurred most often. The majority of the infected feed samples (75%) were contaminated with more than one type of mycotoxin.

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-B(1), aflatoxin-B(2), aflatoxin-G(1) and aflatoxin-G(2)), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B(1), fumonisin-B(2) and fumonisin-B(3)), 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 microL 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 microg kg(-1) to 42.48 microg 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 microg kg(-1)) and fumonisins (fumonisin-B(1) up to 4330 microg kg(-1), fumonisin-B(2) up to 4900 microg kg(-1), and fumonisin-B(3) up to 299 microg kg(-1)) were detected.

Development and validation of a multi-analyte method for the detection of anabolic steroids in bovine urine with liquid chromatography-tandem mass spectrometry.

Detection of anabolic steroids in animal urine samples is currently performed with GC–MS in our lab. However we found that the detection of 17α-trenbolone (17α-TbOH), 4-chloroandrost-4-ene-3,17-dion (CLAD), 16-β-OH-stanozolol (16OHstan) and α- and β-boldenone (α-Bol, β-Bol) was very difficult, if not impossible. Therefore a sensitive, specific and selective qualitative multi-analyte LC–MS–MS method was developed. The LC separation was achieved by using a Symmetry® C18 column and methanol–water–formic acid (54.7–44.7–0.6) as a mobile phase at a flow-rate of 0.3 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode with positive electrospray interface. Validation of the method was done according to draft SANCO/1805/2000 Rev.1 and a CCβ smaller then 1 ng/ml was obtained for each compound.

Development and validation of a QuEChERS based liquid chromatography tandem mass spectrometry method for the determination of multiple mycotoxins in spices.

A reliable and rapid method for the determination of multiple mycotoxins was developed using a QuEChERS (quick, easy, cheap, effective, rugged and safe) based extraction procedure in highly pigmented and complex spice matrices, namely red chilli (Capsicum annum ssp.), black and white pepper (Piper nigrum ssp.). High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used for the quantification and confirmation of 17 chemically diversified mycotoxins. Different extraction procedures were studied and optimized in order to obtain better recoveries. Mycotoxins were extracted from the hydrated spices using acidified acetonitrile (1% formic acid), followed by partitioning with NaCl and anhydrous MgSO4; excluding the use of dispersive-solid phase extraction. Significant matrix effect was compensated using the matrix matched calibration curves. Electrospray ionization at positive mode was applied to simultaneously detect all the mycotoxins in a single run time of 20min. Multiple reaction monitoring mode, choosing at least two abundant fragment ions per analyte was applied. Coefficients of determination obtained were in the range of 0.9844-0.9997. Recoveries (ranging from 75% to 117%) were in accordance with the performance criteria required by the European Commission. Intra-day reproducibility ranged from 4% to 22% for most of the mycotoxins. The limit of quantification ranged from 2.3 to 146μgkg(-1). The validated method was finally applied to screen mycotoxins in ten of each spice matrix. Aflatoxins, ochratoxin, fumonisins, sterigmatocystin and citrinin were among the detected analytes. Positive findings were further confirmed using relative ion intensities. The potentiality of the method to be used for confirmatory purposes according to Commission Decision 2002/657/EC was assessed.

A multi-analyte LC-MS/MS method for the analysis of 23 mycotoxins in different sorghum varieties: the forgotten sample matrix

An LC-MS/MS method was developed and validated for the detection and quantification of 23 mycotoxins in different varieties of sorghum. The method performance characteristics were as follows: suitable linearity ranges for all 23 mycotoxins with p-value >0.05; limits of detection (1.2-50 μg/kg), limits of quantification (2.5-100 μg/kg), repeatability (RSDr, 7-22%), intermediate precision (RSDR, 14-44%) and apparent recovery (0.2-11%, expressed as bias). The method was applied to analyze 10 samples obtained from retail shops in Belgium (n=8) and Germany (n=2). Nine of the 10 samples (90%) were positive for the following mycotoxins: aflatoxin B1 (50 μg/kg), alternariol monomethyl ether (<LOQ - 79 μg/kg), alternariol (303-357 μg/kg), diacetoxyscirpenol (<LOQ - 91 μg/kg), fumonisin B1 (<LOQ - 95 μg/kg), fumonisin B2 (<LOQ), fumonisin B3 (<LOQ), T2 (<LOQ) and zearalenone (<LOQ).

Detection of anabolic steroids in dietary supplements: the added value of an androgen yeast bioassay in parallel with a liquid chromatography-tandem mass spectrometry screening method.

Recently we constructed a recombinant yeast cell that expresses the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. When exposed to testosterone, the concentration where half-maximal activation is reached (EC(50)) was 50 nM. Eighteen different dietary supplements, already analysed by a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the presence of anabolic steroids, were screened for androgenic activity. Eleven samples containing at least one anabolic steroid, with a concentration that was around or above 0.01 mgunit(-1) according to LC-MS/MS, were also positive in the bioassay. Seven samples did not contain any of the 49 compounds screened for in LC-MS/MS. In contrast two of them were positive in the bioassay. Bioassay-directed identification, using the bioassay as an off-line LC-detector and LC-time of flight-MS with accurate mass measurement was carried out in these two samples and revealed the presence of 4-androstene-3beta,17beta-diol and 5alpha-androstane-3beta,17beta-diol in the first and 1-testosterone in the second supplement, showing the added value of the bioassay in comparison with a LC-MS/MS screening method alone.

Investigation into the Possible Natural Occurence of Semicarbazide in Macrobrachium rosenbergii Prawns

In the past year there has been an increased incidence in Belgium of cases of positive semicarbazide (SEM) tests in imported freshwater Macrobrachium rosenbergii prawns, seemingly indicating the possible abuse of nitrofurazone, a banned antimicrobial agent. This was in contrast to all other European countries where no significant increase in SEM-positive samples was detected. A possible explanation for this discrepancy between Belgium and the other European Union member states could be the fact that only in Belgium were whole prawns (meat + shell) analyzed for the presence of tissue-bound metabolites of nitrofurans, whereas in the other countries only the edible part (meat) of these prawns was analyzed. To investigate the possible natural occurrence of SEM in freshwater prawns, an animal trial was set up. In this experiment two groups of 10 juvenile M. rosenbergii, previously raised under standardized laboratory conditions, were stocked into two separate aquaria, a control group under reference conditions (no addition of nitrofurazone) and a group exposed to a daily dose of 50 mg of nitrofurazone L(-1) of culture water. Results of this animal trial proved that SEM naturally occurs in M. rosenbergii prawns but that at the current minimum required performance limit (MRPL) no tissue-bound SEM can be found in the meat of nontreated animals. In addition to this animal trial, commercial samples of other crustacean species, the shell and meat of which were analyzed separately, were also analyzed for the presence of SEM.

Effectiveness of hand sorting, flotation/washing, dehulling and combinations thereof on the decontamination of mycotoxin-contaminated white maize

Maize is one of the major staple foods of Sub-Saharan Africa and is consumed as whole or dehulled grain. In this region, where the environmental conditions favour fungal growth and mycotoxin production, the majority of the population are subsistence consumers who, unfortunately, have little or no access to mycotoxin testing of their food. In an attempt to develop feasible reduction strategies in dietary mycotoxin exposure of the population, a three-factorial design experiment was conducted to examine and compare the efficacy of hand sorting, flotation, dehulling and combinations thereof in removing naturally occurring aflatoxins, fumonisins, nivalenol, deoxynivalenol and alternariol in shelled white maize. Regression analysis was used to determine the significant (p < 0.05) process variables on the removal of mycotoxins from the maize. Results from this experiment indicated that hand sorting had the greatest effect on mycotoxin removal, while flotation yielded the least effect. In particular hand sorting left < 6% of aflatoxin B1 and < 5% of fumonisin B1. Based on these results, hand sorting of maize grains is being recommended as a last line of defence against mycotoxin exposure among subsistence consumers. Graphical Abstract

Implementation of acrylamide mitigation strategies on industrial production of French fries: challenges and pitfalls

This study evaluated various additives or process aids on the industrial production of French fries, based on their acrylamide mitigation potential and other quality parameters. The application of acetic and citric acid, calcium lactate and asparaginase was investigated on the production of frozen par-fried French fries at the beginning and end of the 2008 and 2009 potato storage season. Despite the fact that some of these treatments significantly reduced acrylamide content of the final product in preliminary laboratory experiments, their application on the industrial production of French fries did not result in additional acrylamide reductions compared to the standard product. Asparaginase was additionally tested in a production line of chilled French fries (not par-fried). Since for this product a longer enzyme-substrate contact time is allowed, a total asparagine depletion was observed for the enzyme treated fries after four days of cold storage. French fries upon final frying presented acrylamide contents below the limit of detection (12.5 μg kg⁻¹) with no effects on the sensorial properties of the final product.

Development, validation and application of an ultra high performance liquid chromatographic-tandem mass spectrometric method for the simultaneous detection and quantification of five different classes of veterinary antibiotics in swine manure

In this study, a fast, simple and selective ultra high performance liquid chromatographic-tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous detection and quantification of colistin, sulfadiazine, trimethoprim, doxycycline, oxytetracycline and ceftiofur and for the detection of tylosin A in swine manure was developed and validated. First, a simple extraction procedure with acetonitrile and 6% trichloroacetic acid was carried out. Second, the supernatant was evaporated and the pellet was reconstituted in 1 ml of water/acetonitrile (80/20) and 0.1% formic acid. Extracts were filtered and analyzed by UHPLC-MS/MS on a Kinetex C18 column using gradient elution. The method developed was validated according to the criteria of Commission Decision 2002/657/EC. Recovery percentages varied between 94% and 106%, repeatability percentages were within the range of 1.7-9.2% and the intralaboratory reproducibility varied between 2.8% and 9.3% for all compounds, except for tylosin A for which more variation was observed resulting in a higher measurement uncertainty. The limit of detection and limit of quantification varied between 1.1 and 20.2 and between 3.5 and 67.3 μg/kg, respectively. This method was used to determine the presence and concentration of the seven antibiotic residues in swine manure sampled from ten different manure pits on farms where the selected antibiotics were used. A link was found between the antibiotics used and detected, except for ceftiofur which is injected at low doses and degraded readily in swine manure and was therefore not recovered in any of the samples. To the best of our knowledge, this is the first method available for the simultaneous extraction and quantification of colistin with other antibiotic classes. Additionally, colistin was never extracted from swine manure before. Another innovative aspect of this method is the simultaneous detection and quantification of five different classes of antibiotic residues in swine manure.