Prof. Dr. Sarah De Saeger Laboratory of Food Analysis Ghent University, Belgium

1. Mycotoxins: analysis and human exposure
Prof. Dr. Sarah De Saeger
Laboratory of Food Analysis
Ghent University, Belgium
www.food2know.org www.mytox.be

2. Outline
1.Introduction
2.Overview of analytical methods
1.Confirmatory analysis
2.Rapid screening tests
3.Human exposure to mycotoxins
September 2014

3. 1.Introduction
2.Overview of analytical methods
1.Confirmatory analysis
2.Rapid screening tests
3.Human exposure to mycotoxins
September 2014

4. Mycotoxigenic
Fungi
September 2014

5. Mycotoxins
= secondary fungal metabolites with toxic effects for
humans and animals
Field Storage
Aspergillus √ √
Fusarium √
Penicillium √
Claviceps √
Alternaria √ √
September 2014

6. More than 400 chemically diverse mycotoxins have
been identified.
Ergocornine
Fumonisin B1
Aflatoxin B1
Deoxynivalenol
September 2014 Zearalenone

7. AFLATOXINS:
Tropical climate (recently also found in Southern Europe)
Pre- and/or post-harvest
Carcinogen (group 1 IARC) - liver
Aflatoxin B1
Correlation 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)
Nephrotoxic
Ochratoxin A
Possible carcinogen (group 2B IARC)
Associated to Balkan endemic nephropathy
September 2014

8. CITRININ:
Aspergillus, Penicillium and Monascus
Post-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.
1 Citrinin
September 2014

9. TRICHOTHECENES (DON, T-2):
Moderate/cold climate (also more data from
Africa)
Pre-harvest
Deoxynivalenol
Diarrhea, vomiting, immunotoxic, gastro-intestinal, …
Pigs most sensitive
Cereals, cereal products …
FUMONISINS:
Fumonisin B1
Worldwide
Pre-harvest
Possible carcinogen (groep 2B IARC) – esophagus
Spina bifida (NTD)?
Horses September (ELEM), 2014
pigs (PE)
Maize, cornflakes, polenta …

10. ZEARALENONE:
Worldwide
Pre-harvest
Hyperestrogenism: pigs, sheep, poultry
Maize, maize products …
ERGOT ALKALOIDS:
Worldwide
Pre-harvest (rye, wheat)
Ergotamine, ergocornine, ergosine, ergocryptine …
Interaction with adrenergic, dopaminergic and serotinergic
receptors.
Ergotism; St Anthony’s fire (gangreen)
September 2014

11. ALTERNARIA TOXINS:
Alternaria alternata
Pre-harvest and during storage
In 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

12. 1.Introduction
2.Overview of analytical methods
1.Confirmatory analysis
2.Rapid Screening tests
3.Human exposure to mycotoxins
September 2014

13. 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 mycotoxins
September 2014

14. 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

15. Mycotoxin analysis in food and feed -
General scheme:
1. Sampling = selection of a representative
sample of a given size from a bulk lot
2. Sample preparation = grinding + sub-sampling
3. Analysis
a. Extraction of mycotoxins from the food/feed
b. Clean-up of the extract
c. 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

16. Masked (MODIFIED)
mycotoxins
Rychlik et al, Mycotoxin Research,
2014 ‘Proposal of a comprehensive
definition’
MYCOTOXINS
MASKED MYCOTOXINS
September 2014

17. Quality Assurance
1. Method validation
a. Commission Regulation 2006/401/EC
b. Commission Decision 2002/657/EC (criteria for LC-MS/MS, but only for
feed and animal products)
2. Proficiency Tests
3. Official CEN methods
4. Accreditation according to ISO 17025
September 2014

18. Take home message:
MYCOTOXIN ANALYSIS
I. Chemical diversity
II. Co-contamination
III. Low concentration levels
IV. Different analytical approaches –
general analytical scheme
V. Sampling can be the largest source
of error
VI. Masked mycotoxins
VII. Quality Assurance
September 2014

19. Confirmatory analysis
Confirmation?
Confirmatory method means methods that provide full or
complementary information enabling the substance to be
unequivocally identified and if necessary quantified at the
level of interest.
Commission Decision 2002/657/EC.
September 2014

20. Multi-analyte LC-MS/MS
Liquid Chromatography
tandem mass spectrometry
Advantages:
Measuring more than 25 mycotoxins in one single run
Identification, quantification and confirmation of analytes
Possibility to find mycotoxins in matrices where they were
never expected to be present or found before
Sample extraction and clean-up can be kept very simple: ‘dilute
and shoot’ and ‘evap and shoot’
UPLC provides faster sample troughput and reduced solvent
consumption
Towards multi-contaminant analysis
September 2014

21. 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 sensitivity
Use of (isotope labelled) internal standards
September 2014

22. A typical total ion chromatogram of DON, T2, ZEN and metabolites (2 ng.μL-1)
De Boevre et al. 2012 Food Additives and Contaminants 5 (29): 819-835
September 2014

23. Untargeted high resolution MS
Characteristics:
Measurement of accurate masses
Identification of unknowns
• New 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

24. Multiple stage CID on-line coupling LC with LTQ Ion Trap MS
-C2H2O - H2O
m/z 339 (4): have common fragments with
asparasone A
m/z 315 (3) &
September 2014

25. 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

26. 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

27. General scheme of mycotoxin determination
some samples confirmatory method
Legal limit
many samples, rapid low-cost method
September 2014

28. 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:
1. Non-instrumental (without any special laboratory
equipment) – visual evaluations
‧Good contrast between positive and negative results;
‧Absence of background coloring.
2. Instrumental (simple, handheld, low cost equipment)
September 2014

29. dcELISA icELISA
Signal
2
3
Signal
1
2
1
3
4
Specific anti-mycotoxin antibody
Mycotoxin
Mycotoxin-enzyme conjugate
Mycotoxin-carrier protein
conjugate
Secondary antibody, labeled with
enzyme
Typical
immunoassays
Competitive ELISA principle.
September 2014

30. microtiterplate
ELISA
tube-based
aflatoxin B1; total
aflatoxins
September 2014
corn, corn meal, corn gluten meal, popcorn,
corn/soy blend, soybeans, milled rice,
sorghum, wheat, cottonseed, peanuts, paprika,
chilli
aflatoxin M1 milk and milk products
deoxynivalenol
fumonisin B1; total
fumonisins
zearalenone
T-2 toxin
nuts, cereals and other commodities including
animal feeds
Microtiterplate
ELISA
ochratoxin A cereals, cocoa, coffee, wine
Tube-based
ELISA
aflatoxin corn, cereals, feed, peanuts

31. lateral flow
Membrane tests
flow-through
Gel-based column tests flow-through
Lateral flow aflatoxin B1; total
aflatoxins
September 2014
corn, corn meal, corn gluten meal, popcorn,
corn/soy blend, soybeans, milled rice, sorghum,
wheat
aflatoxin M1 milk
deoxynivalenol wheat, barley
zearalenone corn, cereals, sorghum
Flow-through aflatoxin B1; total
aflatoxins
cereals, soybeans, nuts, derived products
ochratoxin A cereals, wine, green coffee
zearalenone cereals and derived products

32. Lateral Flow Immunoassay (LFD) or
immunochromatographic assay
Anti-mycotoxin antibodies, labeled
with colloidal gold
Mycotoxin
Mycotoxin-carrier protein
conjugate
Secondary antibody
Test line
Sample
pad
Conjugate
pad
Absorbent
pad
Membrane
Control line
Flow
September 2014

33. Advantages of LFD:
1. One-step assay;
2. Use of colloidal gold as label without necessity of
substrate application (contrary to enzymatic assays);
3. Simple dipsticks to more complex systems with plastic
housing;
4. Commercially available for different mycotoxins including
handheld readers;
5. Multi-toxin screening.
September 2014

34. 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

35. Commercially available diagnostic kits:
www.gipsa.usda.gov
www.aoac.org
September 2014

36. 1.Introduction
2.Overview of analytical methods
1.Confirmatory analysis
2.Rapid Screening tests
3.Human exposure to mycotoxins
September 2014

37. 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, α-ZEL, β-ZEL
§ Fumonisins: FB1, HFB1
§ Ochratoxins: OTA, OTα, 4-OH OTA
§SeCptiterimnibner 2014

38. 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 years
Three breastfeeding categories: wholly breastfed, partially breastfed, fully weaned
Exclusion factors: kidney or metabolic disease
Approved by Ethical Committee of Ghent University Hospital
Approved by Ministry of Public Health in Cameroon
September 2014

39. Case study Cameroon: Analytical Procedures (Njumbe Ediage et
al. Anal. Chim. Acta 2012)
Organic phase
Evaporate 40 °C
+ 200 μl H2O/MeOH/FAc
(61,8/37,9/0,3)
15 min centrifuge (14000 x g)
20 μl lower layer LC-MS/MS
10 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 SAX
10 ml MeOH/H2O
(85/15)
10 ml MeOH
Sample
1 ml H2O
5 ml MeOH/FAc (99/1)
+ 500 μl hexane
September 2014

40. Case study Cameroon: Results
September 2014

41. 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

42. Case study Cameroon: Results
§ Significant differences in the mean concentration levels of OTA (p=0.01)
and β-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.
September 2014
§ The mean concentration of the different mycotoxins was statistically the
same across the different age groups (p> 0.05)

43. Case study Belgium: Study Design
September 2012 – January 2014
Approved by Ethical Committee of Ghent University Hospital
Cluster sampling
100 children 300 adults
19 - 65 year
3 -12 year
Flanders - Wallonia - Brussels
Companies/schools
Year variation
25 % adults
Winter 2013-2014
Morning urine vs. 24h
Additional research
September 2014
Funding:
Federal Public Service of Health, Food Chain Safety and Environment (RT11/02 BIOMYCO);

44. Case study Belgium: Study Design
Contact employee
Informed Consent
General questionnaire
Recruitment strategy
Check exclusion factors
Give ID number
Ad random recruitment
Check representativeness
Confirm participation
1 week before sampling
General questionnaire
Food Frequency Questionnaire
Instructions
Urine collection material
1 Sampling day day before sampling
Exclusion factors
Exposure 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

45. Case study Belgium: Results (season 1)
Amount of participants per
city
1 2 3 5
September 2014

46. b Thank you for listening!
September 2014