Alternaria Toxins -Analysis & Occurrence UNDESINSTITUT

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Alternaria Toxins -Analysis & Occurrence

Theresa Zwickel Keith Richards Horst Klaffke

National Reference Laboratory forMycotoxins in Food und Feed

Theresa Zwickel Fachgruppe 83 NRL Mykotoxine Seite 2

Outline

• The mould Alternaria

• The toxins

• Background

• Toxicology

• Our current Alternaria research project at BfR

• Future work at the BfR


Alternaria – an interesting mould speciesDivision: EumycotaClass: Deuteromycetes (Fungi imperfecti)Order: Moniliales (Hyphomycetes)Family: Dermatiaceae (Black mould)

(dark coloured mycelium and/or spores fromMelanine-occlusion)

Genus: Alternaria


Plant pathogenicityAlternaria infestation

Alternaria spp.A. alternata cerealsA. tenuissima vegetables, fruitsA. solani potatoe, tomato, pepperA. brassicae cabbageA. citri citrus fruit

Black mould on wheat earsPhoto: Astrid Oldenburg


9 8

712

1110

6

542

1

3

OOH

OHOH

OH O

H

Altertoxin I (ATX-I)

A B

C

D E

9 8

712

1110

6

542

1

3

OOH

OH

OH O

O

H

Altertoxin II (ATX-II)

9 8

712

1110

6

542

1

3

OHO

OH O

O

H H

Altertoxin III (ATX-III)

5

43

2

NH

CH3

CH2CH2

CH3

OH CH3

O

O

H

Tenuazonsäure (TeA)

N

N

N

NH

CH3

CH3

CH3

OO

O

OCH3

CH3

Tentoxin

12

3

4

O 568

910

7

OOH

O

CH3 OH

CH3

Alternariol-9-methylether (AME)

12

3

4

O 568

910

7

OOH

OH

CH3 OH

Alternariol (AOH)

1

23

4

O 568

910

7

OOH

OH

OHO

CH3

Altenuisol (ALS)

1

23

4

O 568

910

7

OOH

O

OH

CH3

CH3

OH(Iso)-Altenuen(ALT, Iso-ALT)

Alternaria toxins – chemical structures


„Alternaria-Toxins“: a short history

2003BfR statement on current status:

need for research into occurrance and toxicityof Alternaria toxins in food and feedstuffs

2011Statement from EFSA on Alternaria toxins

2012BfR initialise Alternaria toxins research project


EFSA-StatementScientific Opinion on the risks for animal and public health related to thepresence of Alternaria toxins in feed and food1

1. EFSA Journal 2011;9(10):2407 [97 pp.]. www.efsa.europa.eu/en/efsajournal/pub/2407.htm

Non - genotoxic• Tentoxin• Tenuazonic acid

Toxins fall into 2 (roughly categorised) groups:

Genotoxic• Alternariol• Alternariol monomethylether

• Very little toxicological data exists (more needed)• Exposure:no complete data

Toxicological potential TTC


TA in infant cereals in µg/kg (Rychlik et al. TUM; Mycotoxin Workshop 2013 )

Oats & barley & spelt 30 ± 1

Wheat & oats & rye & maize & barley 33 ± 2

Wheat & milk powder & banana 8 ± 0,4

Wheat & milk powder 3 ± 0,1

Sorghum/millet Lot 1 900 ± 40

Lot 2 130 ± 5


Lot 2 900 ± 70


Lot 2 1200 ± 100


Lot 2 380 ± 25


Provisional Risk Assessment by the EFSA (Rychlik et al. TUM)

0

1

2

3

4

5

3 5 4 5 5 5 6 5 7 5 8 5 9 5 10 5 11 5

Aufnah

meTA

[µg/kg

KGproTag]

Körpergewicht [kg]

TTC [1,5 µg/kg KG pro Tag]

Hirsebrei [30 g; 900 µg/kg TA]



Babygläschen [200 g; 15 µg/kg]

Getreidebrei [30 g; 30 µg/kg]

Babytee [200 mL; 2 µg/l TA]

Fencheltee [200 mL;17,5 µg/l TA]

Daily intake of TA depending on consumption and body weight

TTC: threshold of toxicological concern: 1.5 µg/kg bw

30 g sorghum baby cereals(mean 900 µg/kg)

20 g sorghum baby cereals

10 g sorghum baby cereals

baby jar

other cereals

baby tea

fennel teaBodyweight (kg)

Daily intake

(ug/Kg

BW

) per day


Two main approaches for mycotoxin analysis!

Mycotoxinsin food and animal feed

Identification & characterisationof moulds(taxonomy)

Identification & quantificationof mycotoxins

• Mykological approaches• Molecular biological approaches

•Biochemical methods

• Analytical instrumental methods

Avoidance strategies

Exposure evaluation


Clean-Up

Silica SPE columns

Alternaria-Toxin-Multi-MethodI. Sample preparation

Extraction

Modified QuECHERS approach


XIC of -MRM (38 pairs): 195.849/138.700 Da ID: Tenuazonic Acid Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Turbo S... Max. 1.0e4 cps

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0.00

1.00e42.11TEA

XIC of -MRM (38 pairs): 256.999/214.700 Da ID: Alternariol Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Turbo Spray) Max. 1.3e5 cps

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0.0

1.0e510.28

AOH

XIC of -MRM (38 pairs): 290.963/229.200 Da ID: Altenuen Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Turbo Spray) Max. 3.4e4 cps

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0.0

3.4e4 10.36ALT

XIC of -MRM (38 pairs): 413.112/141.100 Da ID: Tentoxin Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Turbo Spray) Max. 2.7e5 cps

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0.0

2.0e510.92

TEN

XIC of -MRM (38 pairs): 271.020/256.000 Da ID: Alternariolmethylether Quant from Sample 13 (Std7_1) of PP_099_Serie 12_24-09-2012.wiff (Tur... Max. 2.2e4 cps

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0.0

2.0e412.38 AME

XIC of -MRM (10 pairs): 413.232/141.000 Da ID: Tentoxin 1 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turbo Spray) Max. 1.4e4 cps

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min

0.0

1.0e45.27

TEN

XIC of -MRM (10 pairs): 256.987/214.600 Da ID: Alternariol 1 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turbo Spray) Max. 1.0e4 cps

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min

0.00

1.00e4 4.64

AOH

XIC of -MRM (10 pairs): 270.942/256.100 Da ID: Alternariolmethylether 1 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turb... Max. 1.2e5 cps

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min

0.00

1.00e55.66

AME

XIC of -MRM (10 pairs): 291.081/229.400 Da ID: Altenuen 1 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turbo Spray) Max. 1.4e4 cps

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min

0.0

1.0e44.96

1.76 ALT

XIC of -MRM (10 pairs): 196.187/111.800 Da ID: Tenuazonic acid 2 from Sample 16 (1S ACN 1P) of PP 115_Serie_1_29-08-2013.wiff (Turbo Spr... Max. 2.1e5 cps

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min

0.0

2.0e54.12

TEA

Alternaria-Toxin-Multi-MethodII. Chromatography

Betterrentention timefor TEA!!!


Multi-Alternaria-Toxin-MethodIII. Matrix Effects – Signal Suppression / Enhancement

XIC of -MRM (10 pairs): 196.187/111.800 Da ID: Tenuazonic acid 2 from Sample 24 (KAL 7 W) of Test Tea_02-09-2013.wiff (Turbo Spray) Max. 5160.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e42.2e4

TEA wheat

TEA maize


Multi-Alternaria-Toxin-MethodIV: Linearity

AME

y = 398.52x + 1125.6R2 = 0.9994

0.00E+00

5.00E+03

1.00E+04

1.50E+04

2.00E+04

2.50E+04

3.00E+04

3.50E+04

4.00E+04

4.50E+04

0 20 40 60 80 100 120

Concentration [ng/ml]

Area

[cps

]

Alternariol methylether matrix matched calibration


Future work on Alternaria toxins at BfR

• Validation of the quantitative Alternaria-toxins analytical multi-method (in-house)• Validation in inter-laboratory studies

• Additional Alternaria-toxins to be added to the method

• Survey of products on the market• to assess the extent of Alternaria-toxins exposure to consumers• risk assessment

Upcoming in November 2013• Inter-laboratory study (with EU-RL, NRLs and NGOs)

• Alternaria toxin standards• Food matrix (tomato juice sample)

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Alternaria Toxins -Analysis & Occurrence UNDESINSTITUT

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