University of Saskatchewan

HARVEST, https://harvest.usask.ca/
College of Agriculture and Bioresources Soils and Crops Workshop

2018-03-06

Establishing LC-MS/MS based

mycotoxin/deoxynivalenol (DON)
platform to support FHB research and
breeding programs

Wang, L.

http://hdl.handle.net/10388/8694
Downloaded from HARVEST, University of Saskatchewan's Repository for Research
 Establishing LC-MS/MS based
mycotoxin/deoxynivalenol (DON)
platform to support FHB research and
breeding programs
Lipu Wang
Cereal & Flax Pathology Lab
Department of Plant Sciences/Crop Development Centre
College of Agriculture and Bioresources

www.agbio.usask.ca
 Fusarium Head Blight (FHB)
• Fungal disease of small grain cereals such as wheat,
barley, oats, rye, and canary seed.
• Fusarium graminearum is the predominant species in
North America.
• Premature bleaching, Fusarium damaged kernels
• Losses in yield, grade and end-use quality

Bleached and shriveled tombstone kernels (left)

compared to healthy wheat kernels.
Photos by Kiersten Wise and Greg Shaner
 Fusarium produces mycotoxins

• Fusarium species produce trichothecene

mycotoxins
• Contribute to plant pathogenesis.
• toxic to humans and livestock.
• Mycotoxin contamination significantly
reduces grain grade and price.
 Trichothecene mycotoxins found in western Canada
• DON is the predominant trichothecene in North America.
• NIV chemotypes prevail in Asia, but was detected in western Canada in 2015.
• Significant shift from the 15-ADON to the more aggressive 3-ADON chemotype
in Western Canada.
• T-2 and HT-2 Toxin are more toxic than DON and found in Western Canada

Deoxynivalenol 3-acetyldeoxynivalenol T-2 Toxin

(DON) (3-ADON)

Nivalenol 15-acetyldeoxynivalenol HT-2 Toxin

(NIV) (15-ADON)
 Masked/modified mycotoxins in grain
• Toxins that are masked from conventional chemical
analysis because their structure has been changed in
plant.
• DON is modified through plant detoxification system
to form less toxic form, Deoxynivalenol-3-glucoside
(D3G) in plant.

DON D3G

Plant
detoxification
 Masked/modified mycotoxins in grain
• Conjugated Mycotoxins might be reversed by gut
microbes in mammalian digestive systems (Gratz et al.,
2013).
• The resistant lines with low DON show a high relative
level of D3G to DON (Amarashinghe et al., 2016).
• Are we breeding variety with low DON due to
converting to D3G?
DON D3G DON

Plant Gut
detoxification Microbes
 Emerging issues in mycotoxin research
• Monitoring chemotypes shift in western Canada
• Identifying types of mycotoxins
• Investigating masked toxins in varieties

requires an analytic tool able to determine these

mycotoxins qualitatively and quantitatively
 LC-/MS/MS (MS2)
The Core Mass Spectrometry Facility
College of Pharmacy and Nutrition

The 4000 QTRAP® LC/MS/MS System is a hybrid triple

quadrupole/linear ion trap mass spectrometer coupled to an
Agilent 1200 quaternary HPLC
PLATFORM I: a state-of-the-art analytical platform
to quantify multiple mycotoxins simultaneously
Analyte Retention Precursor ion Product ions CE (eV)
time RT
(min)

NIV 7.1 371.0 [M+OAc]- 59.0/280.9 42/22

D3G 9.5 517.2 [M+OAc]- 457.2/426.9 20/30

DON 9.8 355.0 [M+OAc]- 295.1/264.9 16/22

3-ADON 12.9 397.0 [M+OAc]- 337.1/307.1 14/22

15-ADON 12.9 397.0 [M+OAc]- 337.0/276.9 12/16

HT-2 15.6 442.2 [M+NH4]+ 263.2/215.5 17/31

T-2 16.3 484.2 [M+NH4]+ 215.2/185.3 29/27

 TIC: from Sample 8 (100) of Nov20.wiff (Turbo Spray) Max. 1.2e6 cps.
T-2
16.26
1.18e6
1.15e6

1.10e6

1.05e6

1.00e6

9.50e5

9.00e5

8.50e5

8.00e5

7.50e5

7.00e5
Intensity, cps

6.50e5

6.00e5

5.50e5

5.00e5

4.50e5
11.58
4.00e5

3.50e5

3.00e5

2.50e5
ADON HT-2
2.00e5 DON 15.60
1.50e5
D3G 9.8
12.93

9.5
1.00e5
NIV
5.00e4
7.1
0.00
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 19.0 20.0
Time, min

LC-ESI-MS/MS TIC of a wheat sample spiked with 7

trichothecenes at a concentration level of 100 ng/mL.
 PLATFORM I: a state-of-the-art analytical platform to
quantify multiple mycotoxins simultaneously
Analyte LOQ (ng/mL) r2
NIV 6.25 0.9999
D-3-G 3.13 0.9984
DON 1.56 0.9973
3AcDON 1.56 0.9973
15AcDON 3.13 0.9996
HT-2 0.16 0.9997
T-2 0.16 0.9995
Limit of quantification (LOQ) and regression coefficient (r2) for each mycotoxin
PLATFORM II: a high throughput DON screening
for breeding programs

Rapid and Easy to operate

High sensitivity
High accuracy
High throughput
Low cost
Rapid One step extraction
LOQ 1.56 ng/mL (ppb)
High-throughput 90 samples per run, 4 runs per day
Low-cost Less 6 CAD per sample

HPLC autosamplers: Large numbers of samples can

be automatically injected onto an HPLC system.
Standard curve of DON from 1.5 ng/mL to 75 ng/mL
with an r2 value of 0.9997 representing good linearity.
 Future direction

• Expand mycotoxin diagnostic library, NX

• Support research studies
• Support breeding programs
• Expand HTP DON assay into other cereal crops
such as barley and canary seed
 Acknowledgements
Project support:
Dr. Randy Kutcher

Project members:
Paulina Cholango Martinez, Mallory Dyck, Gursahib Singh,

Collaborators:
Dr. Anas El-Aneed and Deborah Michel, The Core Mass Spectrometry Facility,
College of Pharmacy and Nutrition
Dr. Yuefeng Ruan, AAFC Swift Current
Drs. Pierre Fobert and Wentao Zhang, ACRD, NRC-Saskatoon

Funding:
the Saskatchewan Agriculture Development Fund (ADF)
the Saskatchewan Wheat Development Commission (SWDC)
Thanks!