screening for fungal and bacterial … for fungal and bacterial metabolies using ... kojic acid 114...
TRANSCRIPT
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
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)