mass spectrometry in food safety · 2016-08-30 · mass spectrometry in food safety paul zavitsanos...
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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
cety
ldeo
xyni
vale
nol
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
cety
ldeo
xyni
vale
nol
Dia
ceto
xysc
irpe
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
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Shanghai AQSIQ Lab
Dr. XiaoJun DENG
Screening 105 Veterinaries in Meat using Agilent 6530 RRLC-QTOF
System
2010-11-4
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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