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