Analytica 2016 Munich, May 11th, 2016

SCREENING FOR FUNGAL AND

BACTERIAL METABOLIES USING

QTRAP® TECHNOLOGY

Michael Sulyok Center for Analytical Chemistry

Department IFA-Tulln

University of Natural Resources and Life Sciences Vienna

Mycotoxins Definition • Low molecular weight-secondary metabolites produced by molds; toxic for

humans and animals

Formation • Aspergillus sp., Penicillium sp., Alternaria sp. Fusarium sp.,

• Usually synthesis of a group of compounds

Function • „Chemical weapons“ against hosts and competitors

Occurrence • 25% of all grains contaminted world-wide (FAO)

• Most important chronic dietary risk-factor

• Economic impact (1.5 bn $ annual loss for US crop production)

→ Regulatory limits

T-2 Toxin Ochratoxin A

(non-UV-absorber) (fluorescent)

HT-2 Toxin Moniliformin Zearalenone Aflatoxin B1

(non-UV-absorber) (ionic) (apolar, fluorescent)

OAcICH3 O

O

H

H H H

H

CH3

H3COH

CH2

OH

O

OOOO

H3C

O

CH2

OHH3C

CH3

H

HHH

H

O

OCH3IOAc

OAc

O

OOO

H3C

O O

(K) O

O

H O

O H

O

O CH 3

O O H

H

H

H

C H 3

H

O

O

H O

H 3 C H

C H 2

O H

OO

OCH3O

O

O

Nivalenol Deoxynivalenol

(very polar) (UV-absorber)

OH O

H

HH

COOH

Cl

O

CH3N O

Major differences:

polarities

UV absorption and fluorescence

ionic nature (dependent on pH)

occurrence in different commodities

concentration levels of interest

O

O

CH3

CH3

CH2

O

O

O

OO

H

HH

H

3

Nature of Mycotoxins

O

O

[COOH][COOH]

O

O [COOH]

[COOH]

CH3 CH3 OH

OH

CH3

OH

NH2

Fumonisins (acidic)

Analysis scheme

Extraction (90 min)

5g sample +

20mL ACN+H2O+HAc

79+20+1 v+v+v

Dilution (1+1) with

ACN/H2O/HAc 20/79/1

Analysis 5 µl of diluted

raw extract injected

without further pre-

treatment

Matrix effects?!

No UPLC?! 2 4 6 8 10 12 14 16 18 20

Time, min 0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

Inte

nsity

, cps

3.23

Agilent 1290 HPLC

Phenomenex Gemini C18, 150x4.6 mm, 5µm

1 mL/min flow rate

+

AB SCIEX QTRAP 5500 in scheduled MRM mode;

2 injections (pos/neg)

710 analytes (May 2016)

0

10

20

30

40

50

60

70

80

90

100

0

25

50

75

100

125

150

175

200

225

250

275

0 5 10 15 20

Re

ve

rse

Ph

as

e G

rad

ien

t

Es

tim

ate

d D

we

ll T

ime

(m

s)

Time (mins)

Estimated Dwell Time for MRM across Time

Excel-Macro provided by Andre Schreiber, Sciex

886 MRMs

1000 ms cycle time

40 s MRM window width

More options (individual

window widths, dwell time

weighting) in the advanced

sMRM mode

-4

-3

-2

-1

0

1

2

3

4

Compilaton of z-scores obtained by our method

in routine proficiency testing (BIPEA)

maize nuts grains animal

feed

baby

food

raisins, pepper,

coffee, milk ….

Case study: Semi-automated production of

sprouted barley as feed for cattle

Diagnosis:

Tremor

Case study: Semi-automated production of

sprouted barley as feed for cattle

Black spots:

Aspergillus sp.?

LC-MS/MS (+) analysis

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 20.0Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

Inte

nsity, c

ps

Kojic acid

114 mg/kg

Fusarinolic acid

8 mg/kg

Fusaric acid

3.8 mg/kg

Cytochalasin E 7 mg/kg

Bikaverin 20 mg/kg

+ Fumonisins (2

mg/kg)

+ Enniatins (200

µg/kg)

+ Tryptoquivaline

derivatives

LC-MS/MS (+) analysis

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 20.0Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

Inte

nsity, c

ps

Kojic acid

114 mg/kg

Fusarinolic acid

8 mg/kg

Fusaric acid

3.8 mg/kg

Cytochalasin E 7 mg/kg

Bikaverin 20 mg/kg

+ Fumonisins (2

mg/kg)

+ Enniatins (200

µg/kg)

+ Tryptoquivaline

derivatives

ESI (-)

Patulin 500 µg/kg

Pseurotin A 900 µg/kg

Patulin, Pseurotin A and Cytochalasin E

confirm A. clavatus

Fusarin C 7.6 mg/kg

The mycotoxin issue in

cocoa

• After freshly harvested pods are

opened, beans undergo natural

fermentation

• Toxigenic fungi may contaminate beans mainly during

fermentation, but also during processing/storage

• Main toxigenic species: A. flavus, A. carbonarius, A.

fumigatus, A. versicolor, A. niger

• Mycotoxin data exist for aflatoxins and OTA only;

usually low-medium prevalence, low concentrations

LC-ESI(+)-MS/MS chromatogram of COG 14

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

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

Inte

nsity, c

ps

2.99

Sterigmatocystin 149 µg/kg

ESI (-):

STERA precursors

Cyclopiazonic acid 246 µg/kg

No AFB1 or

OTA!

Prevalence of selected metabolites

Analyte No. of

positives

Median of

pos. (µg/kg)

Maximum

(µg/kg)

Aflatoxin B1 6 / 57 12.9 270

Ochratoxin A 14 / 57 4.9 11.0

Cyclopiazonic acid 12 / 57 343 4320

Sterigmatocystin 57 / 57 1.86 1910

Averufin 46 / 57 0.89 225

Pseurotin A 39 / 57 123 1890

Nigragillin 57 / 57 n.a. n.a.

Nonactin 48 / 57 1.92 613

• Metabolite pattern suggests that afla- and ochratoxigenic Aspergilli are

less prevalent than A. versicolor, A. fumigatus and A. niger

• Nonactin indicates presence of Streptomyces sp.

• Overall more than 150 different metabolites

Relevance for the consumer?

• Very preliminary data

indicates that

sterigmatocystin may be

present in cocoa and

related products sold in

Austria (4/10 pos)

XIC of -MRM (510 pairs): 367.000/295.100 amu Expected RT: 15.7 ID: Averufin.1 from Sample 1 (AT5-1881-2) of neg15.wiff (Turbo Spray) Max. 7954.0 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 19.0 20.0 Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

7954

Inte

nsity

, cps

15.62

XIC of STERA in

Cocoa Brand B

11.5 µg/kg

Contribution of cocoa and related products to the

overall exposure to sterigmatocystin is not negligible

- 1 / 1259 samples > 11.5 µg/kg (EFSA survey1)

- significant consumption by children2

1 http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/774e.pdf

2 Copetti MV et al., Int J Food Microbiol (2014) 178:13-20

Outlook: Multi-class methods

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

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

Inte

nsity

, cps

Survey in „fufu“

from Nigeria

180 µg/kg

cereulide

Conclusions

• The LC-MS/MS based „dilute-and-shoot“ approach

targeting several hundreds of analytes is sufficiently

accurate (acceptable z-scores in proficiency testing)

• In extreme cases, methods limited to „routine-

mycotoxins“ fail to come up with the correct answer

on the hazards posed by MTs in a particular sample

• Generation of comprehensive data sets on prevalence

of mycotoxins / fungal metabolites feasible

• Identifiation of unexpected exposure scenarios

• Co-occurrence of mycotoxins (and other contaminants)