accurate quantitation of regulated mycotoxins by uhplcby uhplc-ms/ms

Accurate quantitation of regulated mycotoxins by UHPLC MS/MSmycotoxins by UHPLC-MS/MS

Dr. Thomas GlaunerSenior LCMS Applications ChemistAgilent Technologies

In cooperation with:Elisabeth Varga*Rudolf Krska*Rainer Schuhmacher*Franz Berthiller*

*Center for Analytical Chemistry Department of Agrobiotechnology (IFA-Tulln)

October 13, 2011Confidentiality Label

1

Center for Analytical Chemistry, Department of Agrobiotechnology (IFA Tulln), University of Natural Resources and Life Sciences Vienna, Austria

Outline

Mycotoxins

Stable isotope dilution assay (SIDA) for the mycotoxins regulated in Stable isotope dilution assay (SIDA) for the mycotoxins regulated in the European Union

• Reasons• Reasons

• Approaches

• Sample Preparation and Method

• Results

Summary and conclusions


2

MycotoxinsBackground

}g

myces (Greek) = fungustoxicum (Latin) = toxic = Mycotoxin}

low molecular weight, toxic, secondary metabolites of fungi

produced by e.g.:• Fusarium sp., Aspergillus sp., Penicillium sp.

may

r

toxicity: • acute toxic, carcinogenic, mutagenic,

teratogenic, estrogenic by P

rof.

Bür

stm

te atoge c, est oge cand immunotoxic effects

prov

ided


3

MycotoxinsWhy are they an issue?y y >25% of all agricultural commodities are

contaminated with mycotoxins

annual losses of several hundred million tons of food worldwide

annual economical losses: 1 billion USD (US only)(US only)

100+ countries have regulations for the control of mycotoxins in food and feedcontrol of mycotoxins in food and feed


4

MycotoxinsRelevance for food control Notifications concerning mycotoxins

• (RASFF-Annual reports 2002-2008)

1000

1200alert notifications

mycotoxins total

2500

3000information notifications

mycotoxins total

600

800

1000

1500

2000

2500

6 % % %200

400

600

500

1000

1500

3 % 7 % 6 % 9 % 8 % 8 % 10 %

0

200

2002 2003 2004 2005 2006 2007 2008

10 % 39 % 44 % 40 % 40 % 34 % 35 %

0

500

2002 2003 2004 2005 2006 2007 2008


5

Food contaminants onlineFERA FC24 database access Free access to the RASFF alerts and notifications via FC24 database

Registration through Agilent websiteg g g(http://www.chem.agilent.com/en-US/solutions/foodtesting-agriculture/Pages/default.aspx)


6

Accurate quantitation of mycotoxinsReasons

European Commission Regulation (EC) No 1881/2006

• set maximum residue levels (MRL) for mycotoxins• set maximum residue levels (MRL) for mycotoxins

Single target versus multi-target methods

BUTBUT:

Electrospray ionisation (ESI)

• matrix effects hamper accurate mass spectrometric quantification

Quantification of regulatedmycotoxins at a very high degree of accuracy is requiredof accuracy is required


7

Matrix effects in ESI-MS and quantitationApproachespp

Dilution of the sample• method less sensitive

Matrix matched calibration• tedious• differences within one commodity not compensated

ISA

• differences within one commodity not compensated

Standard addition to each sample• more runs• more costs (time and standards)

Internal calibration• similar compounds (ZAN for ZEN)similar compounds (ZAN for ZEN)• deuterium or 13C-labelled compounds• until this year: only single analyte or group analyte IS-addition• usually associated with rather high costs• usually associated with rather high costs


8

Stable Isotope Dilution Assay (SIDA)Aims

Development of a method fulfilling: • covering all regulated mycotoxins in solid food matricescovering all regulated mycotoxins in solid food matrices• providing best possible accuracy• easy to handle• cost effective• cost effective

Stable isotope dilution assay (SIDA) for LC-MS/MS11 t i• 11 mycotoxins

• 13C-labelled compounds as internal standards• validation of the method for maize


9

Multiple Reaction MonitoringPrinciplesp

Quad Mass Filter (MS2)Quad Mass Filter (MS1)

Collision CellCollision cell breaks ion 210 apart

Q3 monitors only characteristic fragments 158 from ion 210 for quant

Q1 lets only target ion 210 pass through

Spectrum with background ions(from ESI)

210

210158

191

ion 210 for quant

158

210210

222

268 280165 150 170 190 210 160190 210

170 210 250 290

165

Chromatogram High background


10

Low background

Sample preparationUniversal extraction procedurep

Sampling grind and homogenize sample + weight‐in

1. Extraction

Centrifugation

acetonitrile:water:formic acid (80:19.9:0.1, v:v:v)60 min at room temperature on a rotary shaker

Centrifugation

2. Extraction acetonitrile:water:formic acid (20:79.9:0.1, v:v:v)30 min at room temperature on a rotary shaker

Centrifugation

Dry Down

+ISTD

Dry Down

Up‐take

Agilent 6490 QqQ


11

Stable Isotope Dilution Assay (SIDA)HPLC method

Agilent 1290 Infinity LC system consisting of:- binary pump

- wellplate sampler

- column compartment

- diode array detector (not used)

HPLC methodSeparation column: ZORBAX Eclipse Plus C-18 RRHD column,

100 x 2.1 mm, 1.8 µm @ 30°CMobile phase: A: 5 mM HCOONH4+ 0.1% formic acid

B: methanol + 5 mM HCOONH4 + 0.1% formic acidB: methanol 5 mM HCOONH4 0.1% formic acidFlow: 0.35 ml/minGradient: 0.00 min 30 % B

0.50 min 30 % B8 00 min 100 % B8.00 min 100 % B9.50 min 100 % B9.60 min 30 % B

Inj.Vol.: 3 µl


12

Stable Isotope Dilution Assay (SIDA)MS methodSpray chamber conditions:Gas temp.: 140°CDry gas: 16 l/miny gNebulizer: 25 psiSheath gas temp: 350°CSheath gas flow: 11 l/min

Positive NegativeCapVoltage: 4000 V 3000 VNozzle voltage 0 V 0 V

Automatic setup of MRM tables based on selected cycle time, retention times and retention time windows for the individual compounds

• Cycle time 400 ms• Interscan delay 3 5 ms• Interscan delay 3.5 ms• Total No. of MRMs 33• Maximum No. Of concurrent MRMs 12• Minimum Dwell time 39.8 ms


13

• Maximum Dwell time 196.5 ms

Dynamic MRM functionalityComparison of MRM and DMRMp

Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4

MRM

Compounds (10/block)

Cycle Time (sec)50 800.5 0.8 1 0.7

100 70

Max Coincident 20 40 40

Dynamic MRM

30Cycle Time (sec) 0.4 0.4 0.4 0.4

• 2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per analyte.


14

p y

Dynamic MRM functionalityDMRM simulation

3x10

1 4251.45

1.4751.5

1.5251.55

1.5751.6

1.6251.65

1.6751.7

1.725 1 1

11.025

1.051.075

1.11.125

1.151.175

1.21.225

1.251.275

1.31.325

1.351.375

1.41.425

ce>>

>

0 5750.6

0.6250.65

0.6750.7

0.7250.75

0.7750.8

0.8250.85

0.8750.9

0.9250.95

0.9751

Abu

ndan

c

0 150.175

0.20.225

0.250.275

0.30.325

0.350.375

0.40.425

0.450.475

0.50.525

0.550.575

8.366

0.050.075

0.10.125

0.15

Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5

Time >>>

ConcurrentCompoundsCompounds

15

Stable Isotope Dilution Assay (SIDA)Chromatogramg due to same MRM transitions baseline separation required for:

• fumonisin B2 and B3 fl t i G1 d 13C fl t i B1

5x10

3 6

3.8 3.87

• aflatoxin G1 and 13C-aflatoxin B1 • aflatoxin G2 and 13C-aflatoxin B2

AFG1

AFB1

2 4

2.6

2.8

3

3.2

3.4

3.6

4.42

4.17

AFB2

AFB1

Spiked maize sample,6490 QqQ

1 2

1.4

1.6

1.8

2

2.2

2.4

6.44

3.58

ZENAFG2

0

0.2

0.4

0.6

0.8

1

1.2

5.405.961.50

3.37 5.55

DON T‐2

FB1FB2

OTAHT‐2

0

Counts vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6


16

Internal calibration in solventAflatoxin B1

Challenging compound due to low MRLs• 0.1 µg/kg in processed cereal based baby food µg g p y• 2 to 12 µg/kg in nuts and cereals

ISTD-10000 [Aflatoxin B1]2x10

8.5

313.1 -> 241.0 Area=2143313.1 -> 285.0 Area=1656


1.8

313.1 -> 241.0 Area=4679313.1 -> 285.0 Area=4066


6

313.1 -> 241.0 Area=15267313.1 -> 285.0 Area=12266

5.5

6

6.5

7

7.5

8

1 1

1.2

1.3

1.4

1.5

1.6

1.7

3 53.75

44.25

4.54.75

55.25

5.55.75

S/N S/N S/N

2

2.5

3

3.5

4

4.5

5

0 4

0.5

0.6

0.7

0.8

0.9

1

1.1

1 251.5

1.752

2.252.5

2.753

3.253.5

74.1(P2P)

204.4(P2P)

532.5(P2P)

4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5

1

1.5

2

4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5

0.1

0.2

0.3

0.4

4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5

0.250.5

0.751

1.25

0.0075 ng/ml 0.0225 ng/ml 0.075 ng/mlOverlay of 4 individual calibrations


17

0.0075 ng/ml 0.0225 ng/ml 0.075 ng/mlacquired within 45 hour worklist.

Internal calibration in solventOchratoxin A

Challenging compound due to low MRLs• 0.5 µg/kg in processed cereal based baby food

Ochratoxin A - 9 Levels, 9 Levels Used, 36 Points, 36 Points Used, 0 QCs

s 1x10 y = 1 592087 * x + 9 491913E-004

ISTD-10000 [Ochratoxin A]2x10 404.1 -> 238.9 Area=181



µg g p y• 3.0 / 5.0 µg/kg in processed / unprocessed cereals• 10.0 µg/kg in dried vine fruit

Rel

ativ

e R

espo

nse 1x10

0 8

0.9

1

1.1

1.2

1.3y = 1.592087 x + 9.491913E-004R^2 = 0.99850604Type:Linear, Origin:Ignore, Weight:1/x

x10

1

1.05

1.1

1.15

1.2

1.25

404.1 238.9 Area 181404.1 -> 102.1 Area=74

x10

1.5

1.6

1.7

1.8

1.9

404.1 238.9 Area 307404.1 -> 102.1 Area=150

x10

2.4

2.6

2.8

3

3.2

404.1 238.9 Area 808404.1 -> 102.1 Area=323

0.3

0.4

0.5

0.6

0.7

0.8

0.75

0.8

0.85

0.9

0.95

1

0 9

1

1.1

1.2

1.3

1.4

1.4

1.6

1.8

2

2.2S/N8.6

(P2P)

S/N13.8(P2P)

S/N25.3(P2P)

Relative Concentration0 1 2 3 4 5 6 7 8

-0.1

0

0.1

0.2

6.2 6.3 6.4 6.5 6.60.5

0.55

0.6

0.65

0.7

6.2 6.3 6.4 6.5 6.60.5

0.6

0.7

0.8

0.9

6.2 6.3 6.4 6.5 6.6

0.6

0.8

1

1.2


18

0.0077 ng/ml 0.0230 ng/ml 0.0765 ng/mlOverlay of 4 individual calibrationsacquired within 45 hour worklist.

Validation of SIDA methodExperimental setup and resultsp p

Linear range (external calibration in solvent)• 4 orders of magnitude for all toxins, 5 orders for Aflatoxins, T-2, and ZEN

Costs• Additional price per IS per sample is between 0.01 to 1.40 €• Price for all 11 IS per sample < 2 00 €• Price for all 11 IS per sample < 2.00 €

Full validation for maize• Maize: - matrix for which most mycotoxins are regulated

- known for matrix effects and matrix interferences• more costs (time and standards)• Spiking with native mycotoxins before extraction• Six concentration levels with 3 replicates• Spiking with 13C-labelled mycotoxins before analysis to compensate matrix

effects in ESIN l l• No sample clean-up


19

Validation of SIDA method in maizeExtraction of spiked blank maize and reference materials

Blank maize sample spiked with native mycotoxins before extraction• includes 10-fold dilution of matrix in the final extract due to extraction procedure

p

Spike_A6 [Aflatoxin B1]3x10

1.3

1.4

313.1 -> 241.0 Area=3455313.1 -> 285.0 Area=2956

Spike_A5 [Aflatoxin B1]3x10

3.8

4

4.2

313.1 -> 241.0 Area=10388313.1 -> 285.0 Area=8941

Spike_A6 [Ochratoxin A]2x10

3 8

4

4.2

4.4

404.1 -> 238.9 Area=972404.1 -> 102.1 Area=382

Spike_A5 [Ochratoxin A]3x10

1.151.2

1.251.3

404.1 -> 238.9 Area=3193404.1 -> 102.1 Area=1021

p

AFB1 OTA

0 8

0.9

1

1.1

1.2

2 4

2.6

2.8

3

3.2

3.4

3.6

2 6

2.8

3

3.2

3.4

3.6

3.8

0.750.8

0.850.9

0.951

1.051.1

1.15

0.4

0.5

0.6

0.7

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

1 4

1.6

1.8

2

2.2

2.4

2.6

0 350.4

0.450.5

0.550.6

0.650.7

0.75

S/N123.7(P2P)

S/N22.3(P2P)

S/N123.9(P2P)

S/N420.9(P2P)

4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5

0.1

0.2

0.3

4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5

0.2

0.4

0.6

0.8

1

6.2 6.3 6.4 6.5 6.6

0.6

0.8

1

1.2

1.4

6.2 6.3 6.4 6.5 6.60.05

0.10.15

0.20.25

0.30.35


20

0.5 µg/kg 1.5 µg/kg 0.5 µg/kg 1.5 µg/kg

Validation of SIDA methodResults – Sample preparationp p p

Extraction efficiency• Determined by spiking of blank samples before extraction• First extraction: 80% acetonitrile content (60 min)

- recovery between 80 and 110% except for FB1 and FB2• Second extraction: 20% acetonitrile content (30 min)

i d i f FB1 d FB2 90%- improved extraction recovery for FB1 and FB2 to approx. 90%

Matrix effects• Signal suppressiong pp

− 50 to 60% aflatoxins− 50% DON

• Signal enhancementg− Fumonisins, HT-2, T2, OTA

• Effectively compensated by ISTD


21

Validation of SIDA methodResults for maize

Analyte LOQin µg/kg

RA** in % ± RSD in %

Aflatoxin B1 0.04 105 6Aflatoxin B1 0.04 105 6

Aflatoxin B2 0.04 100 4

Aflatoxin G1 0.05 101 5

0 24 101 8Aflatoxin G2 0.24 101 8

Deoxynivalenol 2.5 99 9

HT-2 toxin 2.0 98 7

T-2 toxin 0.17 99 6

Ochratoxin A 0.23 93 7

Zearalenone 0 97 103 11Zearalenone 0.97 103 11

Fumonisin B1 2.5 101 10

Fumonisin B2 0.64 88 7


22

** average for triplicate samples and 6 spiking levels

DMRM database for mycotoxinsCustomize your mycotoxin methody y

Multi-mycotoxin method for 242 mycotoxins and other fungal metabolites has been developedp• Validated for different nuts

• Transitions are shortly available as DMRM database

Erythromycin MevastatinRoquefortine C

Fumonisin B1

Dihydrolysergol

FestuclavineGibberelic acid

Actinomycin D

VerrucofortineT‐2toxin

Ochratoxin A

Aflatoxin B1

Kojic acidActinomycin Dtoxin


23

SummaryUHPLC MS/MS th d UHPLC-MS/MS method• Improved chromatographic resolution

Multiple extraction steps• Enhancement of extraction efficiency especially for fumonisins

Dynamic MRM with fast polarity switching• Most abundant ionization mode and maximized dwell times within a single rung

Addition of internal standards after extraction• Compensation for matrix effects• Minimized costsMinimized costs

Apparent recoveries of 88 to 105% for all mycotoxins• Evaluated by extraction of spiked maize samples• Validated by correct quantitation of 18 reference materials covering all toxin groups• Validated by correct quantitation of 18 reference materials covering all toxin groups

Sensitivity suitable for MRLs• Improved sensitivity of G6490 allows to omit sample concentration resulting in easier

handling and improved robustness


24

handling and improved robustness

Questions


25

Validation of SIDA methodMethod characteristics and requirementsq

Analytes Linear rangeng/mL

LOQs (maize)µg/kg

MRLs (EC Reg. No 1881/2006)

Commodities

0 1 processed cereal based baby food

Aflatoxin B1 0.0075 - 74.6 0.04

0.1 processed cereal-based baby food2.0-12

sum of aflatoxins: 4.0-15.0

nuts and cereals

200 processed cereal-based baby foodDeoxynivalenol 0.23 - 225 2.5 200

500 - 1750

p yprocessed / unprocessed cereals,

bread, pasta, breakfast cerealsFumonisin B1 0.075 - 249 2.5 200

8001000 / 4000

processed maize-based baby foodmaize-based breakfast cereals

maize / unprocessed maizeFumonisin B2 0.075 - 251 0.64 1000 / 4000 maize / unprocessed maize2

HT-2 toxin 0.2 - 202 2.0 implementation of MRLs is expected in

the near future

unprocessed cereals and cereal productsT-2 toxin 0.023 - 75.4 0.17

0.5 processed cereal-based baby food

Ochratoxin A 0.023 – 23.0 0.23 3.0 / 5.010.0

15 / 20 / 80

p yprocessed / unprocessed cereals

dried vine fruitspices / liquorice root / extract

Zearalenone 0 076 - 252 1 02050

processed cereal-based baby foodbread biscuits breakfast cereals


26

Zearalenone 0.076 252 1.0 5075-350

bread, biscuits, breakfast cerealsprocessed / unprocessed cereals

accurate quantitation of regulated mycotoxins by uhplcby uhplc-ms/ms

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