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Mycotoxins: analysis and human exposure
Prof. Dr. Sarah De SaegerLaboratory of Food AnalysisGhent University, Belgium
www.food2know.orgwww.mytox.be
Outline
Introduction
Overview of analytical methods
Confirmatory analysis
Rapid screening tests
Human exposure to mycotoxins
September 2014
Introduction
Overview of analytical methods
Confirmatory analysis
Rapid screening tests
Human exposure to mycotoxins
September 2014
Mycotoxigenic FungiSeptember 2014
Mycotoxins
= secondary fungal metabolites with toxic effects for humans and animals
FieldStorageAspergillusFusariumPenicilliumClavicepsAlternaria
September 2014
More than 400 chemically diverse mycotoxins have been identified.
ErgocornineFumonisin B1Aflatoxin B1DeoxynivalenolZearalenone
September 2014
AFLATOXINS:Tropical climate (recently also found in Southern Europe)Pre- and/or post-harvest
Carcinogen (group 1 IARC) - liverCorrelation with human liver cancer!Acute toxicity
B1, B2, G1, G2 in maize, pistachio, Brazil nuts, dried figs, spices M1 in milk
OCHRATOXIN A:Moderate climate (Penicillium) Tropical (Aspergillus)Post-harvest (ex. grapes)
NephrotoxicPossible carcinogen (group 2B IARC)Associated to Balkan endemic nephropathy
Cereals, coffee, wine, beer, pig kidneys
Aflatoxin B1
Ochratoxin ASeptember 2014
CITRININ:Aspergillus, Penicillium and MonascusPost-harvest Nephrotoxic, and weakly genotoxic but carcinogenicity has not been demonstrated; possible synergistic effect with ochratoxin A.
Available occurrence data were not appropriate to carry out a dietary exposure assessment by EFSA.
Stored grains, beans, fruits, fruit and vegetable juices, herbs and spices.It is also an undesirable contaminant in Monascus fermentation products (generally described as red mould rice), which are in use in Asia since many centuries for meat preservation and food colouring.
CitrininSeptember 2014
TRICHOTHECENES (DON, T-2):Moderate/cold climate (also more data fromAfrica)Pre-harvest
Diarrhea, vomiting, immunotoxic, gastro-intestinal, Pigs most sensitiveCereals, cereal products
FUMONISINS:WorldwidePre-harvest
Possible carcinogen (groep 2B IARC) esophagusSpina bifida (NTD)?
Horses (ELEM), pigs (PE)Maize, cornflakes, polenta
Deoxynivalenol
Fumonisin B1September 2014
ZEARALENONE:WorldwidePre-harvestHyperestrogenism: pigs, sheep, poultryMaize, maize products
ERGOT ALKALOIDS:WorldwidePre-harvest (rye, wheat)
Ergotamine, ergocornine, ergosine, ergocryptine
Interaction with adrenergic, dopaminergic and serotinergic receptors.Ergotism; St Anthonys fire (gangreen)
September 2014
ALTERNARIA TOXINS:Alternaria alternataPre-harvest and during storageIn lentils, oil seeds, tomatoes and products, juices, wine, cereals, carrots
Alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN)
Request from EFSA for monitoring food and feed.
September 2014
Introduction
Overview of analytical methods
Confirmatory analysis
Rapid Screening tests
Human exposure to mycotoxins
September 2014
82% of animal feed samples were contaminated with at least one mycotoxin
75% of the infected samples were contaminated with more than one mycotoxin
= Co- contamination by multiple mycotoxinsSeptember 2014
Levels of mycotoxins in food and feed
Part per million (ppm)1 mg/kg = 0.001 g in 1000 g
Part per billion (ppb)1 g/kg = 0.000001 g in 1000 g
September 2014
Mycotoxin analysis in food and feed -General scheme:
Sampling = selection of a representative sample of a given size from a bulk lot
Sample preparation = grinding + sub-sampling
Analysis
Extraction of mycotoxins from the food/feed
Clean-up of the extract
Detection of mycotoxin in the purified extract
The sampling step can be the largest source of error (depending on the food/feed matrix)!!
September 2014
Masked (MODIFIED) mycotoxins
Rychlik et al, Mycotoxin Research, 2014 Proposal of a comprehensive definition
MASKED MYCOTOXINSMYCOTOXINS
September 2014
Quality Assurance
Method validation
Commission Regulation 2006/401/EC
Commission Decision 2002/657/EC (criteria for LC-MS/MS, but only for feed and animal products)
Proficiency Tests
Official CEN methods
Accreditation according to ISO 17025
September 2014
Take home message:MYCOTOXIN ANALYSISChemical diversity
Co-contamination
Low concentration levels
Different analytical approaches general analytical scheme
Sampling can be the largest source of error
Masked mycotoxins
Quality Assurance
September 2014
Confirmatory analysis
Confirmation?
Confirmatory method means methods that provide full or complementary information enabling the substance to beunequivocally identified and if necessary quantified at the level of interest.
Commission Decision 2002/657/EC.
September 2014
Advantages:
Measuring more than 25 mycotoxins in one single runIdentification, quantification and confirmation of analytesPossibility to find mycotoxins in matrices where they were never expected to be present or found beforeSample extraction and clean-up can be kept very simple: dilute and shoot and evap and shootUPLC provides faster sample troughput and reduced solvent consumptionTowards multi-contaminant analysis
Multi-analyte LC-MS/MS
Liquid Chromatography
tandem mass spectrometry
September 2014
Multi-analyte LC-MS/MS
Pitfalls:
Compromise between conflicting different chemical properties of the analytes (extraction ionisation)Matrix effects (ion suppression ion enhancement)In some (or many?) cases clean-up still remains required to reduce matrix effects and increase sensitivityUse of (isotope labelled) internal standards
September 2014
A typical total ion chromatogram of DON, T2, ZEN and metabolites (2 ng.L-1)De Boevre et al. 2012 Food Additives and Contaminants 5 (29): 819-835September 2014
Untargeted high resolution MS
Characteristics:
Measurement of accurate massesIdentification of unknownsNew masked mycotoxins were detected and many more will be
Collection of full scan spectra with possibility to reprocess stored data (= retrospective data analysis)Qualitative and quantitative analysis
September 2014
m/z 339 (4): have common fragments with asparasone Am/z 315 (3) &
-C2H2O - H2OMultiple stage CID on-line coupling LC with LTQ Ion Trap MS
September 2014
Therefore we decided to perform a detailed fragmentation study of the known compound to help us identify the unknowns by comparison.We did this fragmentation study on-line by coupling the LC with an LTQ Ion Trap MS system.This indicated that Asparasone shared common fragments with compounds (3) and (4); starting from the fragment shown here in red with m/z 297.Compound (1) showed the same neutral losses as for asparasone A; the corresponding fragments showing 16 Da differences, that were attributed to an additional Oxygen atom in compound (1).
Rapid screening tests
Screening?
Screening methods are methods that are used to detect the presence of a substance or class of substances at the level of interest. These methods have the capability for a high sample throughput and are used to sift large numbers of samples for potential non-compliant results. They are specifically designed to avoid false compliant results.
Commission Decision 2002/657/EC.
September 2014
Rapid?
Different meanings depending upon the perspective and expectations of the analyst and the context of the analytical environment.
Assays speed should include sample preparation, extraction, isolation of analyte!
To deal with an increasing number of sample matrices and analytes of interest.
September 2014
some samples confirmatory method
many samples, rapid low-cost method
General scheme of mycotoxin determination
Legal limit
September 2014
Immunochemical screening tests
Simple to use:Simple sample extraction; Minimum assay steps; Short assay time; No or minimum toxic solvents; On-site applicability.
Simple to interpret results:Non-instrumental (without any special laboratory equipment) visual evaluations
Good contrast between positive and negative results;
Absence of background coloring.
Instrumental (simple, handheld, low cost equipment)
September 2014
dcELISAicELISACompetitive ELISA principle.
Typical immunoassaysSeptember 2014
microtiterplate
ELISA
tube-based
September 2014
Very complete range of ELISA available on the market; in all possible forms
lateral flow
Membrane tests
flow-through
Gel-based column tests flow-through
September 2014
The availability of lateral flow/flow-through tests is enormously growing but not yet for all toxins. Some of them are not yet performing as they should be. Still improvements necessary.
Lateral Flow Immunoassay (LFD) or immunochromatographic assay
September 2014
Design of a LFD can be simple (dipstick format) or can be more complex.If we look to literature, LFD is the major described rapid test.
Advantages of LFD: One-step assay;
Use of colloidal gold as label without necessity of substrate application (contrary to enzymatic assays);
Simple dipsticks to more complex systems with plastic housing;
Commercially available for different mycotoxins including handheld readers;
Multi-toxin screening.
September 2014
Why is LFD so poplular?
Pitfalls for rapid screening tests:
Very different sample matrices (matrix interference!!);
Low detection limits are needed;
False positives/false negatives (cut-off level or indicator range??);
Limited quality control;
Cross-reactivity to other toxins;
Robustness of on-site test;
Necessity of matrix-matched calibrations?
September 2014
Why is LFD so poplular?
Commercially available diagnostic kits:
www.gipsa.usda.govwww.aoac.org
September 2014
Introduction
Overview of analytical methods
Confirmatory analysis
Rapid Screening tests
Human exposure to mycotoxins
September 2014
Biomarker analysis
Case study Cameroon: Objectives
Biomonitoring of mycotoxin exposure in Cameroon toddlers (1.5 5 years) through assessment of urinary mycotoxin biomarkers
Target analytes: 7 mycotoxins and their potential biomarkers (18 analytes)
Aflatoxins: AFB1, AFB1-N7 Guanine, AFM1
Trichothecenes: DON, DOM, DON-3Glu, T-2, HT-2
Zearalenone: ZEN, ZEN-14Glu, -ZEL, -ZEL
Fumonisins: FB1, HFB1
Ochratoxins: OTA, OT, 4-OH OTA
Citrinin
September 2014
Case study Cameroon: Study Design
Six villages (2 agro-ecological zones, western highland and humid forest with monomodal rainfall): selection based on a previous study
220 toddlers: one child/household
First morning urine samples
Questionnaire (including 24h dietary recall)
Four age groups: 1 - < 2 years; 2 - < 3 years; 3 - < 4 years; > 4 years < 5 yearsThree breastfeeding categories: wholly breastfed, partially breastfed, fully weaned
Exclusion factors: kidney or metabolic disease
Approved by Ethical Committee of Ghent University HospitalApproved by Ministry of Public Health in Cameroon
September 2014
WH: western highlandHFM: humid forest with monomodal rainfall
Case study Cameroon: Analytical Procedures (Njumbe Ediage et al. Anal. Chim. Acta 2012)
Organic phaseEvaporate 40 C+ 200 l H2O/MeOH/FAc(61,8/37,9/0,3)15 min centrifuge (14000 x g)20 l lower layer LC-MS/MS10 ml urine + 15 ml EtOAc/FAc (99/1)30 min shaken + 10 min centrifuge (4000g)water phase+ 0,4 M Na2CO3 (pH 6,5)dilute in MeOH (1/5)SPE SAX10 ml MeOH/H2O (85/15)10 ml MeOHSample1 ml H2O5 ml MeOH/FAc (99/1)
+ 500 l hexane
September 2014
SAX SPE is voor de FUM
Case study Cameroon: Results
September 2014
Seven of the 18 analytes were detected in one or more samples: OTA, DON, AFM1, FB1, ZEN, beta-ZOL, alpha-ZOL. The co-occurrence rate of 2, 3 and 4 co-occurring mycotoxins per sample was 35%, 5% and 5% respectively.
Case study Cameroon: Results
Limit of quantification: 0.02 7.3 ng/mL
160/220 (73%) tested positive
(Njumbe Ediage et al, Environment International, 2013)
September 2014
Seven of the 18 analytes were detected in one or more samples: OTA, DON, AFM1, FB1, ZEN, beta-ZOL, alpha-ZOL. The co-occurrence rate of 2, 3 and 4 co-occurring mycotoxins per sample was 35%, 5% and 5% respectively.
Case study Cameroon: ResultsSignificant differences in the mean concentration levels of OTA (p=0.01) and -ZEL (p= 0.017) between the two agro-ecological zones.
Did not correlate with the food preferences of the different regions
Mean AFM1 concentrations were significantly different across the different weaning categories. There were no differences in the mean OTA (and other mycotoxins) concentrations and weaning categories.
The mean concentration of the different mycotoxins was statistically the same across the different age groups (p> 0.05)
September 2014
Case study Belgium: Study Design
Approved by Ethical Committee of Ghent University Hospital
Cluster sampling100 children300 adults19 - 65 yearFlanders - Wallonia - BrusselsSeptember 2012 January 20143 -12 yearCompanies/schools
Year variation 25 % adultsWinter 2013-2014Morning urine vs. 24hAdditional research
Funding:Federal Public Service of Health, Food Chain Safety and Environment (RT11/02 BIOMYCO);September 2014
Case study Belgium: Study Design
Contact employeeInformed ConsentGeneral questionnaireRecruitment strategy
Check exclusion factors Give ID numberAd random recruitmentCheck representativenessConfirm participation
1 week before sampling
General questionnaireFood Frequency QuestionnaireInstructionsUrine collection material
1 day before samplingSampling day
Exclusion factorsExposure to a large extent of mycotoxins in another way than food-Diseases interfering with metabolism of mycotoxins and creatinin-More than one family member is participating in the study
September 2014
Case study Belgium: Results (season 1)
Amount of participants per city
1 2 3 5
September 2014
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September 2014
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September 2014
September 2014