AOAC/ASFILAB Workshop Paris, November 2009
Potential and limitations of LC-MS/MS based multi-
mycotoxin analysis
Michael Sulyok, Rainer Schuhmacher, Rudolf Krska
AOAC/ASFILAB Workshop Paris, November 2009
Outline
Introduction – Mycotoxins
Setting up an LC-MS/MS multi-mycotoxin method
Pitfalls and counteractive measures
Benefits
AOAC/ASFILAB Workshop Paris, November 2009
MycotoxinsDefinition• Low molecular weight-secondary metabolites produced by molds
(plants; processed food; damp buildings)
Function• „Chemical weapons“ against hosts and competitors
Formation• Fusarium sp., Aspergillus sp., Penicillium sp., Alternaria sp.
• Usually synthesis of a group of compounds
Occurrence
25 % of all cereals are contaminated with mycotoxins
=> most important chronic dietary risk factor (> synthetic
contaminants, pesticides, food additves, plant toxins)
Economic losses: 109 $ / a in US
FAO
AOAC/ASFILAB Workshop Paris, November 2009
Analytes – Structures (I)
OH
O
CH3
H
O
OOH
Zearalenon
O
H
CH2OH
OH
CH3
OH
H
H
H
HO
O
CH3
DON
Trichothecenes
NIV, DON, FUS-X, 3ADON, DAS, HT2, T2,
DON-3-Glucoside, Deepoxy-DON, Verrucarol, Neosolaniol,
Monoacteoxyscirpenol, Verrucarin
gastrointresinaldisturbance, immunos., inhibition of proteinsynthesis
Zearalenone
derivatives
ZON, ZOLs,
ZON-Sulfat, ZON-Glucoside
xenoestrogen
O O
O
O O
OCH3
Aflatoxins
Aflatoxin B1, B2, G1, G2
potent carcinogen; hepato-toxic (2004: 350 death
cases in Kenya)
Aflatoxin B1
O
CH
2
CH N
OOOH
Cl
O
O
HH
CH3
Ochratoxins
Ochratoxin A, B, α
nephrotoxin; teratogen,cancerogen,
immunosuppr.Ochratoxin A
AOAC/ASFILAB Workshop Paris, November 2009
Analytes – Structures (II)
ON
O
N
NO
O
OO
O O
O
Enniatins (5)
Beauvericin
Enniatin A, A1, B, B1
APOLAR
cationophor, cytotoxic
Beauvericin
O
O
[COOH][COOH]
O
O [COOH]
[COOH]
CH3
CH3
OH
OH
CH3
OH
NH2
Fumonisins (3)
Fumonisin FB1, FB2, hydrolyzed FB1
HYDROPHILIC, ACIDIC
cancerogen; interfereswith sphingolipid
synthesis
FB1
Ergot alkaloids (4)
Ergotamin, Ergocornin, Ergovalin, Dihydroergosin
POLAR, BASIC
Ergotism
N
NH
CH3
CH3
H
H
AgroclavinOH O
OMoniliformin
pKa: 1.7
cardiotoxic
AOAC/ASFILAB Workshop Paris, November 2009
Multi-analyte methods for mycotoxins:
The reasons...
• 300-400 substances recognized as mycotoxins
13 addressed by legislation
approx. 25 covered by methods described in the literature
• Screening of wide range of analytes necessary for evaluation of
synergistic effects of different mycotoxins (McKean et al., J. Appl.
Toxicol. 2006, 26, 139)
• Unified method for various analytical purposes
Reference method for development of rapid tests
Characterization of standards
Chemotaxonomy – Determination of production strains, metabolite pattern in real-world samples
AOAC/ASFILAB Workshop Paris, November 2009
...and the challenges
range of relevant concentrationsEU: 2 µg / kg (AFB1) –
1500 µg / kg (DON)
chemical diversity of analytes
ionic ⇔ apolar
acidic ⇔ basic
various matrices
(wheat, maize, cereals, wine, milk, …)
sensitivity, linearity selectivity
general detection principle minimum samplepreparation
AOAC/ASFILAB Workshop Paris, November 2009
...and the challenges
range of relevant concentrationsEU: 2 µg / kg (AFB1) –
1500 µg / kg (DON)
chemical diversity of analytes
ionic ⇔ apolar
acidic ⇔ basic
various matrices
(wheat, maize, cereals, wine, milk, …)
sensitivity, linearity selectivity
general detection principle minimum samplepreparation
HPLC / MS-MS analysis of crude extracts
is thisfeasible??
signalsuppression?!
AOAC/ASFILAB Workshop Paris, November 2009
Outline
Introduction – Mycotoxins
Setting up an LC-MS/MS multi-mycotoxin method
Pitfalls and counteractive measures
Benefits
AOAC/ASFILAB Workshop Paris, November 2009
LC-MS/MS – Instrumentation
HPLC: Agilent 1100; Gemini C18
MeOH/H2O gradient, 1% HAc, 5mM NH4Ac
MS: QTrap 4000 MS/MS (equipped withTurboIon Source)
Ion path
Collision cell (N2) ExitSkimmer
Orifice
Q0 Q1 Q2 Q3
From: James W. Hager, MDS Sciex,
ASMS 2002
Ions
Q3 = Trap
Multiplier
Quantification in the SRM-mode (triple quad configuration):
Sequential scanning of fragmentation reactions (2 per analyte, 100ms each); parameter optimization via direct infusion of the analytes
Confirmation of positive results by Enhanced Product Ion scans (trapconfiguration)
AOAC/ASFILAB Workshop Paris, November 2009
Optimization of HPLC conditions
2 4 6 8 12 14 16 18 20Time, min
5000
1.0e4
1.5e4
Intensity, cps
FB1
HFB1
FB2
OHNH
2
OH OH
OH
OH
O
O
O
O
CH3
CH3
OH
OH
CH3
OH
NH2
HOOC
COOH
COOH
COOH
� Acidic eluent for fumonisins necessary
Elution of fumonisins at neutral pH
AOAC/ASFILAB Workshop Paris, November 2009
Elution of fumonisins at pH 3
13.24
2 4 6 8 12 14 16 18 20 Time, min
1e4
2e4
3e4
4e4
Intensity, cps
HFB1
FB1
FB2
� Gemini C18-column
�Linear gradient from
10% to 97% MeOH
�acidified eluent
(1% HAc besides
5mM NH4Ac)
Final HPLC conditions
AOAC/ASFILAB Workshop Paris, November 2009
Separation of ergotamine epimers
11.05 11.10 11.15 11.20 11.25 11.30 11.35 11.40 11.45 11.50 11.55 11.60 11.65 11.70 11.75 11.80 11.85Time, 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
2.1e4
2.2e4
2.3e4
2.4e4
Inte
nsity
, cp
s
11.57
11.71
Quantifier SRM
Qualifier SRM (x5)
Separation of epimersincomplete under acidicconditions; alkalineconditions would be
preferable (∆rt = 2.6 min)
ergotamine
ergotaminine
AOAC/ASFILAB Workshop Paris, November 20092 4 6 8 10 12 14 16 18 20
Time, min0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
Inte
nsity
, cps
15.16
17.30
10.927.98
15.416.70
Scheduled MRM vs. periods : A useful software upgrade
2 4 6 8 10 12 14 16 18 20Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
Inte
nsity
, cps
12.16
9.207.43
9.32 12.5613.37
8.62
15.86
8.0611.39
15.029.506.13
16.08
Positive run 1
Positive run 2
Periods:
SRMs of defined setsof analytes arescanned during fixedperiods
• demands extremelystable retention times
• difficulties to setperiod limits in multi-analyte methods
(max. 100)
• a lot of junk data is
produced (waste of acquisition time)
AOAC/ASFILAB Workshop Paris, November 2009
Merged ESI (+) Scheduled SRM chromatogramof 125 analytes
2 4 6 8 10 12 14 16 18 20Time, 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
• Separate time window for each analyte (tR ± 0.4 min)
⇒ minor retention shiftsare irrelevant
⇒ optimum use of acquisition time (SRM dwell times generateddynamically)
⇒ more analytes can beincluded in a single run(300-1000); 2 runs/sample
instead of 3
AOAC/ASFILAB Workshop Paris, November 2009
Choice of solvent for extraction of wheat
Best compromise: Low water content, low pH ⇒ ACN/H2O/HAc 79/20/1(results similar for maize)„acidified acetonitril/water mixtures are the default method for multi-analyte
analysis..“ (Mol et al., Anal. Chem. 2008, 9450-9458)
0
20
40
60
80
100
120
HT2
-Toxi
n
Niv
alen
ol
Zeara
lenon
Och
rato
xin
A
Afla
toxi
n B1
Ergoc
ornin
Monili
form
inFum
onisi
n B2
ex
tra
cti
on
eff
icie
nc
y (
%)
MeOH/H2O 1/1 ACN/H2O 84/16 ACN/H2O/HAc 79/20/1
(n=3);
0.5g sample +
2ml solvent;
Extract
diluted 1:10
AOAC/ASFILAB Workshop Paris, November 2009
Sample preparation scheme
Grinding
Extraction (90 min)
5g sample +
20mL ACN+H2O+HAc
79+20+1 v+v+v
(optimized!)
Dilution (1+1) withACN/H2O/HAc 20/79/1
Analysis 2x21 mins.
(pos. / neg. mode)
Reduced time and material consumption!
AOAC/ASFILAB Workshop Paris, November 2009
Outline
Introduction – Mycotoxins
Setting up an LC-MS/MS multi-mycotoxin method
Pitfalls and counteractive measures
Benefits
AOAC/ASFILAB Workshop Paris, November 2009
Matrix effects – the foe of LC-MS/MS
• Not to be exchanged with interference – effect is invisible
• Effect caused by co-eluting matrix constituents
competition for electrical charge
modification of surface tension of ESI droplet
• Extent is both matrix- and analyte-dependent
• May effect only small sections of chromatogramm
⇒ Use of non-coeluting internal standards insufficient; closely elutinganalogues with similar structure (zearalanon vs. zearalenon) may beacceptable
Approaches: Standard addition
Matrix matched calibration
Isotopically labelled internal standards
AOAC/ASFILAB Workshop Paris, November 2009
Extraction efficiency ⇔⇔⇔⇔ matrix effects (1)
13.7 13.8 13.9 14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 15.0 15.1 15.2 15.3 15.4 15.5Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4378
Inte
nsity
, cps
14.02
14.03
liquid standard
Peak area: 29000
spikedbreadcrumbs
Peak area: 22700
O
O
OH
OH
RAlternariol R=OH
Alternariolmethylether R=OCH3
⇒ Apparent recovery: ca.
80%
Signal suppression or
bad extraction efficiency?
AOAC/ASFILAB Workshop Paris, November 2009
Extraction efficiency ⇔⇔⇔⇔ matrix effects (2)
13.7 13.8 13.9 14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 15.0 15.1 15.2 15.3 15.4 15.5Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4378
Inte
nsity
, cps
14.02
14.03
14.01
liquid standard
Peak area: 29000
spikedbreadcrumbs
Peak area: 22700
spiked breadcrumbextract
Peak area: 22900
O
O
OH
OH
RAlternariol R=OH
Alternariolmethylether R=OCH3
⇒ Extraction efficiency not
affected
Signal suppression (ca
20%)
AOAC/ASFILAB Workshop Paris, November 2009
169.000
14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 15.0 15.1 15.2
Time, min
0.0
1000.0
5000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
Inten
sity, cps
14.57
liquid standard
100 µg/kg
Blank breadcrumbs
spiked with 100 µg/kgCitrinin
O
O
O OH
OH
51.00014.6
Blank breadcrumbs
Raw extract spiked
164.000
⇒ No signal suppression
Extraction efficiency ca. 30%
Extraction efficiency ⇔⇔⇔⇔ matrix effects (3)
AOAC/ASFILAB Workshop Paris, November 2009
Method Validation
A: liquid standard; 10
concentration levels, 4 injections each
B: blank extract;
spiked at 10 concentration levels
C: spiked samples;10
levels, each in triplicate; extractionand dilution
Calibration functions(1/x weighted)
Comparison of slopes
C / A: apparent recovery
B / A: signal suppression
/ enhancement
C / B: recovery of the
extraction step
Process standard deviation
Precision
Trueness
AOAC/ASFILAB Workshop Paris, November 2009
Validation of maize
75124932.2Moniliformin
93-10778-108; 141Z4G79-101; 141Z4G0.5-0.7;3Z4SZearalenons
86-10396-100; 83OTαααα98-100; 71OTαααα0.4-1.2Ochratoxins
57-75101-104;63 HFB147-700.4-1.4Fumonisins
86-103101-10994-1060.6-1.9Beau + Ennis
86-11027-6230-620.6-1.3Ergot alkal.
95-11048-62; 18AFB149-68; 17AFB11.3-2.8Aflatoxins
66(NIV, D3G) - 9992-110; 157D3G93-106; 74NIV0.3-1.3B-Trichos
97-10873-9276-980.2-0.8A-Trichos
Recovery of extraction (%)
Signal supp. / enh. (%)
Apparentrecovery (%)
ProcessSTDEV (%)
Analyte class
• matrix effects for Aflas, Ergots and some other compounds
• incomplete extraction of polar analytes
• excellent reproducibility and linearity ⇒⇒⇒⇒ matrix calibration to correct
for matrix effects (variation between individual samples?!)
AOAC/ASFILAB Workshop Paris, November 2009
Validation of rice
51113582.2Moniliformin
83 - 10599 - 10989 - 1100.8-2.6Zearalenons
95 - 97; 62 OTαααα99 - 11496 - 98; 71 OTαααα0.8-1.9Ochratoxins
74 - 85100 - 10774 - 881.2-2.7Fumonisins
1011001012.0Beauvericin
94 - 10695 - 10693 - 1121.6-3.2Ergot alkal.
93 - 10982 - 10194 - 983.1-5.1Aflatoxins
89 - 10197 - 10688 - 1050.9-4.0B-Trichos
96 - 10499 - 10598 - 1050.5-0.7;A-Trichos
Recovery of extraction (%)
Signal suppr. / enh. (%)
Apparentrecovery (%)
Processstdev (%)
Analyte class
Process st.dev. < 6.0% in spelt and barley (Z4S: 12%)
Signal suppr./enh.: spelt: Aflas 75-91%, Ergots 70-99%
barley: Aflas 47-75 %, Ergots 66-89%, ZONs: 52-69%
⇒⇒⇒⇒ Method transferable to other grain matrices, matrix-MATCHED calibration
AOAC/ASFILAB Workshop Paris, November 2009
Variation of signal intensities within a given matrix
Maize: Variation of analytical signal acceptably low (exception: D3G 32%)
Rice: Dramatically decreased signal intensities for some analytes in red rice(compared to peeled and brown rice)
⇒⇒⇒⇒ For a quantitative analysis, the model matrix must match the samples as close as possible!!!
Blank extracts of 3
individual samplesspiked at 5 concentration levels
0
5
10
15
20
25
30
35
A-
Trichos
B-
Trichos
Aflas Ergots Ennis Fumos Ochras ZONs Moni
RS
D (
%)
maize
rice
AOAC/ASFILAB Workshop Paris, November 2009
Analysis of DON in reference materialswith and without 13C15-DON
0
100
200
300
400
500
600
700
1 2
DO
N [
µg
/kg
]
CRM maize CRM wheat
(certified) target
concentrations13C labeled internalstandards compensatematrix effects even on less robust instruments(QTrap 2000)
Approach applicable to
regulated toxins, but not for multi-mycotoxin analysis(costs, availabilitiy)
AOAC/ASFILAB Workshop Paris, November 2009
ESI-MS/MS-Parameters
Contributionof Dr. Robert Köppen,
BAM, Berlin
AOAC/ASFILAB Workshop Paris, November 2009
Confirmation of positive results in HPLC-MS/MS according to 2002/657/EC
• Identification points: 1 IP parent ion, 1.5 IP product ion
• 3 IPs required for mycotoxins (group B of Annex I in 96/23/EC)
• 4 IPs required for banned substances
• 2 SRM transitions per analyte
• Retention time must agree with authentic standard (± 2.5% rel.)
• Intensity ratio of 2 SRMs must agree with authentic standard (± 20% rel.)
• Complied by most methods, but...
...is this sufficient?!
AOAC/ASFILAB Workshop Paris, November 2009
Roridin A in citrus fruits?
13.65 13.70 13.75 13.80 13.85 13.90 13.95 14.00 14.05 14.10 14.15Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
Inte
nsity
, cps
13.89
SRM 550.4/249.2
SRM 550.4/231.2
Comparison to standard
∆rt: 0.09 min (0.7%)macrocyclictrichothecene produced by Myrothecium roridum
and Myrothecium
verrucaria
O
O
O
OO
O
O
OH
OH
H H
H
AOAC/ASFILAB Workshop Paris, November 2009
Comparison of product ion spectra of the precursour ion m/z=550.4
Inte
nsity
, cp
sIn
tensity
, cp
s
58.wiff (Turbo Spray) Max. 1,7e5 cps.
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
m/z , amu
1,0e4
2,0e4
3,0e4
4,0e4
5,0e4
6,0e4
7,0e4
8,0e4
9,0e4
1,0e5
1,1e5
1,2e5
1,3e5
1,4e5
1,5e5
1,6e5
1,7e5249,2
133,1
231,2
550,4
333,2
137,1
533,3
213,2203,2 385,3185,2247,1
161,2403,2
195,2159,1131,1
201,1119,1 469,0
337,3219,2147,1187,1175,3 344,8 490,1 515,2
379,2105,3 331,1207,2 387,6
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
m/z , amu
249,2133,1
231,2550,4
333,2
137,1533,3
213,2203,2 385,3185,2247,1
161,2403,2
195,2159,1131,1
201,1119,1 469,0
337,3219,2147,1187,1175,3 344,8 490,1 515,2
379,2105,3 331,1207,2 387,6
reference
standard
substanceeluting at 14.05 min in orange
369,2
533,3
411,2
550,3
515,3
455,3
413,3393,2
473,3437,2367,3
341,2
429,2161,0 409,2351,2
385,3395,3
431,3469,2375,1365,2187,1 315,2295,2 333,2261,2 383,2245,2231,1
133,0 401,2373,0 487,3159,1 497,2197,1 269,2287,2265,3 329,2247,1177,2 445,4217,2 453,4381,3119,2137,0 534,2207,1 489,5361,3169,0 425,2 509,4
+EPI (550,40) CE (25): 14,024 to 14,128 min from Sample 1 (z1_056) of 56.wiff (Turbo Spray) Max. 1,6e5 cps.
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z, amu
1,0e4
2,0e4
3,0e4
4,0e4
5,0e4
6,0e4
7,0e4
8,0e4
9,0e4
1,0e5
1,1e5
1,2e5
1,3e5
1,4e5
1,5e5
1,6e5
+EPI (550,40) CE (25): 14,024 to 14,128 min from Sample 1 (z1_056) of 56.wiff (Turbo Spray) Max. 1,6e5 cps.
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600m/z, amu
1,0e4
2,0e4
3,0e4
4,0e4
5,0e4
6,0e4
7,0e4
8,0e4
9,0e4
1,0e5
1,1e5
1,2e5
1,3e5
1,4e5
1,5e5
1,6e5369,2
533,3
411,2
550,3
515,3
455,3
413,3393,2
473,3437,2367,3
341,2
429,2161,0 409,2351,2
385,3395,3
431,3469,2375,1365,2187,1 315,2295,2 333,2 383,2249,2231,1
133,0 401,2373,0 487,3159,1 497,2197,1 269,2287,2265,3 329,2247,1177,2 445,4217,2 453,4381,3119,2137,0 534,2207,1 489,5361,3169,0 425,2 509,4
substance eluting
at 14.05 min in citrus fruits
⇒ Two SRM transitions may be still insufficient for confirmation of identity!
AOAC/ASFILAB Workshop Paris, November 2009
Outline
Introduction – Mycotoxins
Setting up an LC-MS/MS multi-mycotoxin method
Pitfalls and counteractive measures
Benefits
AOAC/ASFILAB Workshop Paris, November 2009
Identification of emerging mycotoxins
• 49 different analytes identified in the 247 sub-samples
AOAC/ASFILAB Workshop Paris, November 2009
Identification of emerging mycotoxins
• 49 different analytes identified in the 247 sub-samples
• regulated toxins: DON 21st in ranking
AOAC/ASFILAB Workshop Paris, November 2009
Identification of emerging mycotoxins
• 49 different analytes identified in the 247 sub-samples
• regulated toxins: DON 21st in ranking
AOAC/ASFILAB Workshop Paris, November 2009
Identification of emerging mycotoxins
• 49 different analytes identified in the 247 sub-samples
• regulated toxins: DON 21st in ranking
AOAC/ASFILAB Workshop Paris, November 2009
Identification of emerging mycotoxins
• 49 different analytes identified in the 247 sub-samples
• regulated toxins: DON 21st in ranking
AOAC/ASFILAB Workshop Paris, November 2009
Identification of „unexpected“ analyte/matrix combinations
TIC of +EPI (329.10) CE (45) CES (20): from Sample 1 (epiafm1_peanutcont8_008) of 8.wiff (Turbo Spray) Max. 7.1e6 cps.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time, min
0.0
5.0e5
1.0e6
1.5e6
2.0e6
2.5e6
3.0e6
3.5e6
4.0e6
4.5e6
5.0e6
5.5e6
6.0e6
6.5e6
7.0e6
Inte
nsity
, cps
15.28
15.46
12.80
16.36
17.96
14.4314.21
11.509.52 12.21
4.029.96
8.18
„milk toxin“ in peanuts???
EPI scan for Aflatoxin M1 in contaminated peanut
AOAC/ASFILAB Workshop Paris, November 2009
Identification of „unexpected“ analyte/matrix combinations
+EPI (329.10) CE (45) CES (20): 11.449 to 11.551 min from Sample 1 (epiafm1_peanutcont8_008) of 8.wiff (Turbo Spray)Max. 1.2e5 cps.
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350m/z, amu
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
1.05e5
1.10e5
1.15e5
1.19e5
Inte
nsity
, cps
329.2
273.2
135.1259.2247.1229.1153.2
301.1 315.0181.2 283.2117.1 203.0 328.4213.1111.0 332.8
100 120 140 160 180 200 220 240 260 280 300 320 340m/z, amu
329.1
273.1
229.1 259.1247.1 301.1
153.2 181.2 283.2
Presence of Aflatoxin M1 in
peanut confirmed!
Isolated Aspergillus
flavus strain
produces AFM1 (1/100 of AFB1)
EPI-spectrum of
referencestandard
AOAC/ASFILAB Workshop Paris, November 2009
XIC of +MRM (298 pairs): 143.0/113.2 amu Expected RT: 3.4 ID: Kojic acid from Sample 1 (hazelnut6_016) of 14.wiff (Turbo Spray)
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
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6291
Inte
nsity
, cps
3.19
3.56 18.272.95
„Self-validation“
AFB1
AFG1
AOAC/ASFILAB Workshop Paris, November 2009
XIC of +MRM (298 pairs): 143.0/113.2 amu Expected RT: 3.4 ID: Kojic acid from Sample 1 (hazelnut6_016) of 14.wiff (Turbo Spray)
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
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6291
Inte
nsity
, cps
3.19
3.56 18.272.95
„Self-validation“
AFB1
AFG1Kojic acid
Aspergillus flavus
AOAC/ASFILAB Workshop Paris, November 2009
XIC of -MRM (100 pairs): 118.0/46.0 amu Expected RT: 3.3 ID: 3-NPA from Sample 1 (hazelnut6_016) of 48.wiff (Turbo Spray)
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
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
Inte
nsity
, cps
3.11
„Self-validation“
3-NPA (A.
flavus)
Alternariol
Alternariol-methylether
AOAC/ASFILAB Workshop Paris, November 2009
XIC of -MRM (100 pairs): 118.0/46.0 amu Expected RT: 3.3 ID: 3-NPA from Sample 1 (hazelnut6_016) of 48.wiff (Turbo Spray)
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
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
Inte
nsity
, cps
3.11
„Self-validation“
3-NPA (A.
flavus)
Tenuazonic
acid
Alternariol
Alternariol-methylether
MacrosporinA
Alternaria
sp.!
AOAC/ASFILAB Workshop Paris, November 2009
Consistency of metabolite production
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Time, min
1.0e5
2.0e5
4.0e5
5.0e5
Inte
nsity
, cp
s
14.07
15.8011.17
13.70 15.989.17.68
7.68 Chanoclavin
5 µg/kg
9.1Festuclavin
2 µg/kg
11.17Meleagrin2600 µg/kg
14.07 Fumonisin B233000 µg/kg
15.80 Enniatin B25 µg/kg13.70
Mycophenolicacid 760 µg/kg
ESI(-)-MS/MS:
Emodin 2500 µg/kg
Alternariol methylether
7 µg/kg
AOAC/ASFILAB Workshop Paris, November 2009
Fumonisin B2 in dark bread
100 150 200 250 300 350 400 450 500 550 600 650 700 750m/z, amu
2,0e6
4,0e6
6,0e6
8,0e6
Inte
nsity
, cp
s
336,4
318,4
354,4
512,3688,5
706,5670,3530,4
494,6149,2 372,4238,3 548,3
100 150 200 250 300 350 400 450 500 550 600 650 700 750m/z, amu
336,3
318,4
354,4
512,3 706,5688,4
494,3 530,5372,3301,3 670,3
548,6
Standard
100 150 200 250 300 350 400 450 500 550 600 650 700 750m/z, amu
2,0e6
4,0e6
6,0e6
8,0e6
Inte
nsity
, cp
s
336,4
318,4
354,4
512,3688,5
706,5670,3530,4
494,6149,2 372,4238,3 548,3
100 150 200 250 300 350 400 450 500 550 600 650 700 750m/z, amu
336,3
318,4
354,4
512,3 706,5688,4
494,3 530,5372,3301,3 670,3
548,6
Standard
Specific production of FB2 by Aspergillus niger
(Frisvad et al. 2007)
EPI spectra of m/z=706.3 at 14.14 min
black spotof moldydark bread
⇒ confirmation of occurrence of unusualtoxin patterns (no FB1 or
FB3)
AOAC/ASFILAB Workshop Paris, November 2009
7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
4.2e4
4.4e4
4.6e4
4.8e4
4.9e4
Inte
nsity
, cps
Isofumigaclavine
Fumigaclavine
Penicillic acid
Festuclavine
Pentoxyfylline
Viridicatin
Enniatins
Alamethicin F30
Puromycin
Myriocin
Nonactin
Monactin
Dinactin
Valinomycin
Multi-metabolite analysis in settled dust from a waste-management plant
⇒⇒⇒⇒ Simplicity of method allowssimple transfer
to othermatrices and metabolites
AOAC/ASFILAB Workshop Paris, November 2009
Conclusions
Pros and Cons of LC-MS/MS multi-mycotoxin analysis:
Use in routine analysis is limited by matrix effects⇒⇒⇒⇒ fast screening analysis (followed by dedicated quantitative method)
⇒⇒⇒⇒ 13C labelled internal standards may overcome this problem (costs?)
Method is a valuable tool at the cutting edge of science⇒⇒⇒⇒ gives a more comprehensive picture of human exposure to toxicfungal metabolites (emerging mycotoxins, unusual toxin / matrixcombinations, including non-food matrices)
⇒⇒⇒⇒ chemotaxonomy – metabolite production in real world samples
⇒⇒⇒⇒ method is flexibe enough to include more analytes and matricese.g. study co-exposure to fungi and bacteria at the metabolic level