potential and limitations of lc-ms/ms based multi- mycotoxin...

AOAC/ASFILAB Workshop Paris, November 2009

Potential and limitations of LC-MS/MS based multi-

mycotoxin analysis

Michael Sulyok, Rainer Schuhmacher, Rudolf Krska

Outline

Introduction – Mycotoxins

Setting up an LC-MS/MS multi-mycotoxin method

Pitfalls and counteractive measures

Benefits

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

Analytes – Structures (I)

Zearalenon

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

Aflatoxins

Aflatoxin B1, B2, G1, G2

potent carcinogen; hepato-toxic (2004: 350 death

cases in Kenya)

Aflatoxin B1

Ochratoxins

Ochratoxin A, B, α

nephrotoxin; teratogen,cancerogen,

immunosuppr.Ochratoxin A

Analytes – Structures (II)

Enniatins (5)

Beauvericin

Enniatin A, A1, B, B1

APOLAR

cationophor, cytotoxic

Beauvericin

[COOH][COOH]

O [COOH]

[COOH]

Fumonisins (3)

Fumonisin FB1, FB2, hydrolyzed FB1

HYDROPHILIC, ACIDIC

cancerogen; interfereswith sphingolipid

synthesis

Ergot alkaloids (4)

Ergotamin, Ergocornin, Ergovalin, Dihydroergosin

POLAR, BASIC

Ergotism

AgroclavinOH O

OMoniliformin

pKa: 1.7

cardiotoxic

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

...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

...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?!

Outline

Benefits

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

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)

Optimization of HPLC conditions

2 4 6 8 12 14 16 18 20Time, min

Intensity, cps

� Acidic eluent for fumonisins necessary

Elution of fumonisins at neutral pH

Elution of fumonisins at pH 3

2 4 6 8 12 14 16 18 20 Time, min

Intensity, cps

� Gemini C18-column

�Linear gradient from

10% to 97% MeOH

�acidified eluent

(1% HAc besides

5mM NH4Ac)

Final HPLC conditions

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

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

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

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

9.207.43

9.32 12.5613.37

8.0611.39

15.029.506.13

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)

Merged ESI (+) Scheduled SRM chromatogramof 125 analytes

2 4 6 8 10 12 14 16 18 20Time, min

• 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

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)

Monili

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

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!

Outline

Benefits

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

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

liquid standard

Peak area: 29000

spikedbreadcrumbs

Peak area: 22700

RAlternariol R=OH

Alternariolmethylether R=OCH3

⇒ Apparent recovery: ca.

Signal suppression or

bad extraction efficiency?

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

liquid standard

Peak area: 29000

spikedbreadcrumbs

Peak area: 22700

spiked breadcrumbextract

Peak area: 22900

RAlternariol R=OH

Alternariolmethylether R=OCH3

⇒ Extraction efficiency not

affected

Signal suppression (ca

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

1000.0

5000.0

sity, cps

liquid standard

100 µg/kg

Blank breadcrumbs

spiked with 100 µg/kgCitrinin

51.00014.6

Blank breadcrumbs

Raw extract spiked

164.000

⇒ No signal suppression

Extraction efficiency ca. 30%

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

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?!)

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

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

Trichos

Aflas Ergots Ennis Fumos Ochras ZONs Moni

Analysis of DON in reference materialswith and without 13C15-DON

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)

ESI-MS/MS-Parameters

Contributionof Dr. Robert Köppen,

BAM, Berlin

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?!

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

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

Comparison of product ion spectra of the precursour ion m/z=550.4

tensity

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

1,7e5249,2

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

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

413,3393,2

473,3437,2367,3

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

+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

1,6e5369,2

413,3393,2

473,3437,2367,3

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!

Outline

Benefits

Identification of emerging mycotoxins

• 49 different analytes identified in the 247 sub-samples

• regulated toxins: DON 21st in ranking

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

14.4314.21

11.509.52 12.21

4.029.96

„milk toxin“ in peanuts???

EPI scan for Aflatoxin M1 in contaminated peanut

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

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

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

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

3.56 18.272.95

„Self-validation“

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

3.56 18.272.95

AFG1Kojic acid

Aspergillus flavus

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

5000.0

3-NPA (A.

flavus)

Alternariol

Alternariol-methylether

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

5000.0

3-NPA (A.

flavus)

Tenuazonic

Alternariol

Alternariol-methylether

MacrosporinA

Alternaria

Consistency of metabolite production

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Time, min

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

Fumonisin B2 in dark bread

100 150 200 250 300 350 400 450 500 550 600 650 700 750m/z, amu

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

512,3 706,5688,4

494,3 530,5372,3301,3 670,3

Standard

100 150 200 250 300 350 400 450 500 550 600 650 700 750m/z, amu

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

512,3 706,5688,4

494,3 530,5372,3301,3 670,3

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

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

2000.0

4000.0

6000.0

8000.0

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

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

potential and limitations of lc-ms/ms based multi- mycotoxin...

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