Mass Spectrometryin Food Safety

Paul ZavitsanosGlobal Food Safety Marketing Manager

Agilent’s Lab-bench to Lab-bench

Market Development Model

Create local research/technical development teams in Europe, the Americas, China, and India

Identify key chemical and biological applications (unmet needs)

Collaborate with respected researchers to make these applications a reality

Support researchers world wide on specific applications

Communicate our findings worldwide

Commercialize successful applications

Existing Core Chemical Applications GC/MS, LC/MS, GC/QQQ, LC/QQQ, LC/Q-TOF, LC/TOF, ICP/MS

Veterinary drugs• Identification/quantification of antibiotics, steroids, and

other growth hormones

The challenge: increase speed and sensitivity while decreasing cost

Pesticides • Quantification of known pesticides• Identification/quantification of new pesticides and metabolites

Trace metals • Identification/quantification of elements such as lead and mercury

Emerging Chemical Applications

Steroid hormones (difficult to measure through conventional techniques) • GC/QQQ – Dramatic success in 2010 Winter Olympics

Mycotoxins (over 3,000 known; immediate toxic effects)• Typically LC/MS

Dioxins in food (traditionally requires high-res equipment/specialized skills)• GC/QQQ – Fast, simple, more sensitive than conventional GC approaches

Marine toxins (small-molecule compounds produced by shellfish)• LC/QQQ (quantification of known targets) • LC/TOF (discovery of unknowns)

Allergens (chemical aspects to identification) • LC/MS, LC/Q-TOF, GC/QQQ – Small-molecule allergens

Emerging Biological Applications

Species identification (accidental contamination vs. intentional fraud)• PCR, bioanalyzer, specialty bioreagents – Identify biological compounds at theDNA level; Manipulate genetic information

Allergens (biological aspects to identification)• LC/MS, LC/Q-TOF – Intact protein discovery with bio-confirm software can determine the exact sequence of molecule in question

• Measurement by biological reagents

Pathogen identification (serotype)• Mass Codes PCR with MS detection • Identification at the DNA level• DNA manipulation • Tag and recognize dangerous bacteria• Serotype determination – At what point in manufacturing process iscontamination taking place?

DISCOVERY of unknown chemical containments

TOF techniques • Visualize and differentiate

compounds that were previously“hidden” under one peak

• Modern TOF instruments: 10x greater mass discrimination. Can determine empirical formulabased on the mass of thecompound in question.

Novel data mining techniques (such as DRS, MFE, PCD)• Discover more compounds/

contaminants, faster, and with greater precision

• Data review and processing: weeks vs. minutes

MYCOTOXINS

Determination of Aflatoxins (B1, B2, G1, G2) in Food Matrices using Triple Quadrupole LC/MS/MS

Experimental Work and Data provided by:

Yang Chen and Jack CappozzoNational Center for Food Safety and Technology

Presented by Peter Stone, Agilent Technologies, Santa Clara, CA.

Chromatography

B1

G2

B2G1

aflatoxins

Overlaid EICsQuant ion & 2 x Qualifiers

(1ppb)

Isotopically labelled aflatoxins

Overlaid EICsQuant ion & Qualifier ion

(2.5ppb)

B1

G2

B2G1

Spiked Peanut Samples – Recovery Studies(% Recovery, ± RSD, N=7)

No Internal Standard:

Internal Standard:

Aflatoxin Peanut spikedat 5 ng/g

C18 clean-up

Peanut spikedat 25 ng/g

C18 clean-up

Peanut spikedat 5 ng/g

MycoSep#226

Peanut spikedat 25 ng/g

Mycosep#226B1 96.7 ± 3.4 97.0 ± 4.6 112.0 ± 8.4 104.9 ± 1.7B2 98.3 ± 4.7 97.4 ± 2.9 108.0 ± 4.6 104.5 ± 2.0G1 95.0 ± 5.6 95.0 ± 4.9 109.9 ± 2.1 105.7 ± 3.4G2 100.0 ± 2.3 100.0 ± 2.0 114.7 ± 3.2 106.3 ± 1.1

Aflatoxin Peanut spikedat 5 ng/g

C18 clean-up

Peanut spikedat 25 ng/g

C18 clean-up

Peanut spikedat 5 ng/g

MycoSep#226

Peanut spikedat 25 ng/g

Mycosep#226B1 101.8 ± 3.6 96.1 ± 2.0 100.0 ± 6.8 103.0 ± 3.5B2 102.5 ± 5.5 100.2 ± 5.0 99.4 ± 4.1 102.9 ± 2.7G1 105.7 ± 7.3 99.2 ± 2.2 105.2 ± 4.3 101.7 ± 5.2G2 107.5 ± 10.9 104.9 ± 6.7 109.3 ± 8.7 102.4 ± 3.1

Acknowledgements:• Experimental Work and Data provided by:

• Yang Chen and Jack Cappozzo• (National Center for Food Safety and Technology, IIT)

Photo: Rima Juskelis, Jianwen Xu, Katie Banaszewski, Haoshi Feng, Niranjen Kalle, Fadwa Al-Taher, Jack Cappozzo, Yang Chen

MycotoxinScreening by

LC/QQQ

Dr. Robert VoyksnerDr. Jennifer VoyksnerLC/MS Limited, NC. USA

Comparison of pos and neg ESI LC/MS/MS analysis of 31 mycotoxinsConditions: poroshell 120 2.1x50 mm 2.7 um particles gradient 5-95% ACN 12 min(0.025% TFA for pos esi, 20 mM am

Act neg esi), 0.3 ml/min

Positive ESI MRM transitions

Negative ESI MRM transitions

Recovery for 31 Mycotoxins from spiked corn using 3 different LC/MS/MS gradient analysis conditions

Conditions: 1 g corn spiked with 20-160 ng/g of each mycotoxins, extracted with 2 ml of 80% ACN in water with 0.025% TFA for 10 min, centrifuged and filtered (30K MWCO filter), then diluted with 1.5 ml of water and 10 ul injected onto the LC/MS/MS. Separation used poroshell 120 2.1x50 mm 2.7 um particles gradient 5-95% ACN in 3, 12 and 28 min, 0.3 ml/min, ESI pos ion detection used 0.025% TFA and ESI neg ion detection used 20 mM AM Act.

Analysis of mycotoxins in food

Agilent 1290 Infinity HPLC / G6460A QQQ system

Page 15

Dr. Thomas GlaunerLC-MS Food Application ScientistEuropean Food Group

0.5 g cereal sample

(ground and homogenized)

Sample preparation

Page 16

M. Sulyok, F. Berthiller, R. Kruska, R. Schuhmacher, Rapid Commun Mass Spectrom 2006, 20(18): 2649-2659

Extraction with 2 ml solvent

(CH3CN/H2O/HAc 79 + 20 + 1)

Shaking (90 min) and centrifugation

(2 min @ 3000 Umin-1)

Dilution (0.5 ml sample + 0.5 ml solvent)

(CH3CN/H2O/HAc 20 + 79 + 1)

LC-MS/MS

Page 17

Mycotoxins acquired in negative ion mode

Niv

alen

ol

Deo

xyni

vale

nol

Fusa

reno

n-X

3-A

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xyni

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Zear

alen

on

In negative mode most abundant precursor ions ofmycotoxins have been [M-H]- and [M+COOH-]-,respectively.

Page 18

Mycotoxins acquired in positive ion mode

Neo

sola

niol

Afla

toxi

n B2

HT2

-Tox

in

15-A

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xyni

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nol

Dia

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nol

Afla

toxi

n B1

T2-T

oxin

Fum

onis

in B

2

Fum

onis

in B

1

Fum

onis

in B

3

In positive mode most abundant precursor ions ofmycotoxins have been [M+H]+, [M+NH4

+]+, and[M+Na+]+, respectively.

PLANT HORMONES

Plant Growth Regulators(PGRs)

Page 20

• Plant hormones or Phytohormones are chemicals which regulates plant growth

• Manmade compounds are called PGRs and basically used to regulate growth of cultivated plants, weeds and in vitro grown plants and plant cells

• Major five classes of plant hormones

1-Abscisic acid

2-Auxins

3-Cytokinins

4-Ethylene

5-Gibberellins

• Other known hormones are like salicylic acid,Jasmonates,Plant peptide hormones,Polyamines,Nitric oxide,Strigolactones and Karikins

Sample clean up for PGRs quantitation

1g of homogenized grapes sample

Spiked with mixture of STD PGRs(final cone 5ppb in sample and 50ppb in blank)

Sample extracted in 40ml Methanol Water (50:50)30 min/ambient/shaking

1.0 ml supernatant transferred to 1.7 ml centrifuge tube and

Centrifuge @ 14,000 rpm, 5min

Methodology Sample Preparation – continued.

Solid phase dispersiveClean-up

0.4ml supernatant diluted with 0.6mlMethanol for LC/MS

Vortex for 1 min & centrifuge @ 14,000 rpm, 3min

Add 200mg of C18 ODS SPE bulkSorbent, Agilent (p/n 5982-1182)

0.8 ml supernatant transferred to1.7ml micro-centrifuge tubes

Chlormequat

Zeatin

Kinetin

6-Benzyladenine

IAA

IBA

Ferchlorfenuron

Paclobutrazole

Sample study

Sample spiked with STD (5ppb)

Response for 0.5ppb STD Chlormequat

DIOXINS, FURANS and PCBs

April 2010

Chris SandyEMEA GC-MS Food Segment Scientist

Agilent Technologies UK

Determination of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzo-p-furans (PCDFs) in Foodstuffs using the 7000B Tandem Quadrupole GC-

MS/MS System

Acknowledgements

Dr Karl Petitt, Ms Karolina SonginMarchwood Scientific Services, UK

Prof. Peter Fuerst, Dr Thorsten BernsmannCVUA-MEL, Munster, Germany

Native PCDD and PCDF Isomers

Tetra Penta Hexa Hepta Octa

2378-TCDD in Poultry Egg (1.66 pg TEQ /g Fat)

2378-TCDD in Beef Sample (0.28 pg TEQ /g Fat)

Difference between GC-HRMS results and GC-MS/MS resultsTEQ

Source : CVUA-MEL

PCDD/F-TEQ pg/g

LOD animal feed: GC-HRMS: 0.01-0.06 pg/g; GC-MS/MS: 0.02-0.08 pg/g

(Upper bound)

Difference between GC-HRMS results and GC-MS/MS resultsTEQ ~< 3 pg/g

LOD animal feed: GC-HRMS: 0.01-0.06 pg/g; GC-MS/MS: 0.02-0.08 pg/g

Poultry meat

PCDD/F-TEQ pg/g

MLAL

MLAL

Organic Hen’s eggs from Germany May 2010Hens fed on feed manufactured in Holland

from maize imported from Ukraine

AL ML

(Upper bound)

Dioxins / DBFs Total TEQ* for Bovine meat extract

*TEQ = Toxic Equivalent Concentration Total TEQ = Sum of analytical results for 17 Dx/DBFs, expressed as pg-TEQ/g Fat

Analyte Analytical result TEF Value TEQ pg/g Fat pg/g Fat

Dioxins 2378-TCDD 0.281 1 0.28112378-PCDD 0.237 1 0.237

123478-HxCDD 0.001 0.1 0.0001123678-HxCDD 0.575 0.1 0.0575123789-HxCDD 0.040 0.1 0.004

1234678-HpCDD 0.663 0.01 0.00663OCDD 0.209 0.0001 0.000021

Dioxins Total TEQ pg/g fat 0.586

Furans 2378-TCDF 0.037 0.1 0.003712378-PCDF 0.009 0.05 0.0004523478-PCDF 3.289 0.5 1.6445

123478-HxCDF 1.475 0.1 0.1475123678-HxCDF 1.539 0.1 0.1539234678-HxCDF 1.584 0.1 0.1584123789-HxCDF 0.002 0.1 0.0002

1234678-HpCDF 0.439 0.01 0.004391234789-HpCDF 0.108 0.01 0.00108

OCDF 0.356 0.0001 0.000036Furans Total TEQ

pg/g fat 2.114

Total (Dioxins + Furans)TEQ pg/g fat 2.70

Max permitted level TEQ pg/g fat 3

ANABOLIC STEROIDSand other

VETERINARY DRUGS

Application of the Agilent 7000B to the Determination of Anabolic Steroids in Veterinary Samples

Dr Bruno Le BizecSchool of Veterinary MedicineNantes, France

Agilent 7000 TQ /LABERCAGROWTH PROMOTERS IN TISSUE - DETECTION AND IDENTIFICATION OF ANABOLIC

STEROIDS BY GAS CHROMATOGRAPHY COUPLED TO TANDEM MASS SPECTROMETRY

Molecules Type Transition 1 Collision T1 (eV) Transition 2 Collision T2

(eV) TR (min)*

Trenbolone fraction

17a-trenbolone AR 380.3>323.3 20 449.3>307.3 20 16.00

17ß-trenbolone-d2 EI 444.3>309.3 20 16.15

17ß-trenbolone AR 380.3>323.3 20 442.3>295.3 25 16.17

Molecules Type Transition 1

Collision T1 (eV)

Transition 2

Collision T2 (eV) TR (min)*

Estradiol-zeranol fraction

17a-estradiol AR 416.3>285.2 12 416.3>129.1 15 14.78

17ß-estradiol-d3 EI 419.3>285.2 12 15.17

17ß-estradiol AR 416.3>285.2 12 416.3>129.1 15 15.20

Norgestrel EE 456.3>301.3 25 16.98

Zeranol-d4 EI 437.3>295.2 25 16.57

Zeranol AR 433.3>295.2 25 523.4>433.3 25 16.60

Taleranol-d4 EI 437.3>295.2 25 16.73

Taleranol AR 433.3>295.2 25 523.4>433.3 25 16.76

Some anabolic steroïds detected

Molecules Type Transition 1 Collision T1 (eV) Transition 2 Collision T2

(eV) TR (min)* Faq

Fraction androgènes-progestagènes

17a-nandrolone AR 418.3>194.1 20 418.3>182.1 15 14.45 1

17a-testosterone AR 432.3>209.2 18 432.3>247.2 18 14.82 1

17ß-nandrolone-d3 EI 421.3>194.1 20 14.88 1

17ß-nandrolone AR 418.3>194.1 20 418.3>182.1 15 14.90 1

17ß-testosterone-d2 EI 434.3>211.2 15 15.32 1

17ß-testosterone AR 432.3>209.2 15 432.3>247.2 25 15.33 1

Methyltestosterone-d3 EI 449.3>301.3 25 16.24 2

Methyltestosterone AR 446.3>301.3 20 446.3>340.9 15 16.27 2

Progesterone AR 458.3>157.1 25 443.3>157.1 15 17.53 et 17.86 2

Megestrol-d3 EI 561.4>224.2 25 18.60 3

Megestrol AR 558.4>236.2 25 558.4>453.3 25 18.63 3

Melengestrol-d3 EI 573.4>73.0 25 18.84 3

Melengestrol AR 570.4>73.0 25 570.4>243.2 25 18.86 3

Medroxyprogesterone-d3 EI 563.4>333.3 25 18.90 3

Medroxyprogesterone AR 560.4>315.3 25 560.4>328.3 25 18.92 3

Chlormadinone AR 578.4>143.1 25 578.4>473.3 25 21.03 3

Chromatographic conditions: column 30m 0.25µm 0.25 mm, splitless mode

Good repeatability onTissue NTd3 10 injections

Nortestosterone d3 Concentration 0.5 ppb

Good repeatability onTissue NTd3 after10 injections

more

Nortestosterone d3

COMPOUND DISCOVERY APPROACHES

2010-11-4

44

Shanghai AQSIQ Lab

Dr. XiaoJun DENG

Screening 105 Veterinaries in Meat using Agilent 6530 RRLC-QTOF

System

2010-11-4

45

Results— TIC of Standards and real samples

Standards （5ng/mL)

Meat sample (5ug/kg)

2010-11-4 45

2010-11-4

462010-11-4 46

Result— EIC

标品5ng/mL

样品5ug/kg

2010-11-4

47

Error distribution plot of matrix sample

-8.00

-7.00

-6.00

-5.00

-4.00

-3.00

-2.00

-1.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Erro

r (p

pm

)

Compound

-2ppm < 92% compounds < 2ppm

2010-11-4 47

Results—Accurate Mass

PATHOGEN IDENTIFICATION

MultiMaTCH™ Genotyping(applied to food safety)

Greg RichmondMolecular Preparation Team Agilent Laboratories

Confidentiality Label

DETECTION OF 58 DIFFERENT MASS TAGS BY APCI-MS

Qiagen Extraction of Contaminated Tomato Culture

Instagene Extraction of Contaminated Tomato Culture