Chemical analysis and screening approaches in food safety ... · Chemical analysis and screening approaches in food safety control [email protected]

Chemical analysis and screening approaches in food safety control

[email protected]

Outline

Setting the scene

Chemical screening methods:

1000+ analytes and more in one analysis

Sample preparation

Instrumental measurement

Data analysis: target, semi-non-target, non-target

No time to waste: screening in seconds

Conclusions

Food chain & potential food safety issues

pesticides mycotoxins plant toxins

veterinary drugs

dioxins, PCBs, PAHs PFCs, BFRs

marine biotoxins

processing contaminants

packaging contaminants

spoilage aging

disinfectants, additives, dyes,

micro organisms

adulterants

unknowns

heavy metals radionuclides

nanoparticles

GMO

allergens

Food safety in the media....




Climate change (?): Increased prevalence vibrio bacteria?

Mussels and oysters contaminated with tetrodotoxin (TTX)

Press release Dutch Food Safety Authority (NVWA)

Cyclic economy: mineral oil/ink from recycled packaging: MOSH: mineral oil saturated hydrocarbons MOAH: mineral oil aromatic hydrocarbons

NGO: Dutch retail products contaminated with mineral oil




Impact of internet sales...

Current issues in the EU

RASFF notifications 2012-2015: 1. Organic small molecules: residues, (natural) contaminants... 2. Pathogenic micro-organisms

Hazard category 2012-2015

pathogenic microorganisms 2893

mycotoxins 1810

pesticides 1739

heavy metals 1062

composition 722

food additives/flavourings 503

foreign bodies 468

adulteration/fraud 444

migration 424

allergens 374

vet drugs 313

GMO / novel food 294

non pathotenic microorganisms 281

industrial contaminants 242

biocontaminants 177

organoleptic aspects 164

parasitic infestations 117

radiation 108

packaging issues 97

labelling issues 92

biotoxins (other) 87

feed additives 58

TSEs (e.g. BSE) 53

chemical contaminantion (other) 21

Prevention: production protocols (e.g. GAP, GMP, HACCP, ..) Verification: Analysis of samples throughout the food chain

Food safety: prevention & verification

Research institutes Academia

enforcement of established MRLs

ensure compliance with MRLs due diligence / reputation / image / sales

data for risk assessment (monitoring/surveys) fate studies (processing, animal transfer) emerging risks & unknown toxicants

Governmental labs

Contract labs

Industry labs

The analytical challenge

Residues >1000 Pesticide

>400 Veterinary drugs

Natural contaminants >500 Mycotoxins > 500 Plant toxins

>100 Marine biotoxins

> 1000 Environmental contaminants dioxins, PCB, PAH, flame retardants, PFCs

biocides, anti-fouling, endocrine disruptors, heavy metals

>> 100s Processing contaminants & adulterants acryl amide, heterocyclic amines, furan, 3-MCPD, disinfectants,

packaging contaminants (badge, ESBO, SEM, phthalates, Sn) marker substances (spoilage, irradiation), dyes, melamine

Low levels: 0.001-10 mg/kg

http://www.shutterstock.com/pic-3348361-beautiful-cut-of-prime-rib-beef-roast.html

http://www.shutterstock.com/pic-2505150-milk-carton-ready-for-your-text-vector-illustration.html

http://www.shutterstock.com/pic-3586926-eggs-against-white-background.html

Inorganic Radio nuclides Elements, nanoparticles Organic small molecules Pesticide residues Veterinary drugs / growth promotors Environmental contaminants Natural toxins Processing and packaging contaminants Organic other Proteins Nanoparticles Food composition

Chemical analysis techniques

α, β, γ-spectroscopy AAS, AES, (LC-)ICP-MS

Chromatography UV, Flu, Mass spectrometry

Electrophoresis, Chromatography, MALDI SEM, ...

Profiling/finger prints: NIR, Raman, NMR,......

Evolution in food safety analyses

Traditional

Emerging

Untargeted measurement

LC-full scan HRMS

New approach

Targeted sample prep/measurement

Past SRM/group methods

OPP GC-FPD

PYR GC-ECD OCP

GC-ECD

CARB LC-Flu

ONP GC-MS TBZ

LC-UV

X method Y

decide what you want to know, then analyse

analyse, then decide what you want to know

1-20

A method B F

method G

20-150 analytes/method

Present MRMs

pesticides GC-MS/MS

pesticides LC-MS/MS

PCBs/PAH GC-MS/MS

vet drugs LC-MS/MS

mycotoxins LC-MS/MS

Chemical screening

Wide scope screening methods

Automated detection of 1000+ food toxicants by 1 analysis method

Sample preparation: generic extraction

Aim: Find the best extraction solvent: High extraction yield for ‘all’ food toxicants Low co-extraction of food matrix (proteins, fat, carbohydrates,..)

Generic extraction: initial toxicants tested

Insecticides, fungicides, herbicides (incl. acids) mycotoxins plant toxins

Acephate Clomazone Fenamiphos-sulfone Imazamethabenz-methyl Oxydemeton-methyl Seneciphylline-N-oxide

Aclonifen Cyanazin Fenamiphos-sulfoxide Imidacloprid Paclobutrazole Senkirkine

Aflatoxin B1 Cymoxanil Fenchlorazole-ethyl Iodosulfuron-methyl (-) Parathion-ethyl Simazin

Aflatoxin B2 D, 2,4 (-) Fenhexamid Iprodion Penconazole Solanin, alpha-

Aflatoxin G1 Demeton-S-methyl Fenitrothion Iprovalicarb Pencycuron Spinosyn A

Aflatoxin G2 Demeton-S-methylsulfone Fenoxaprop-p-ethyl Isoproturon Phorate Spinosyn D

Aldicarb Deoxynivalenol (DON) Fenoxycarb Isoxaben Phorate-sulfone Spiroxamine

Aldicarb-sulfone Deoxynivalenol, 15-O-acetyl-4 Fenpropimorph Linuron Phorate-sulfoxide Sterigmatocystin

Aldicarb-sulfoxide Deoxynivalenol, 3-acetyl Fenpyroxymate Lufenuron Phosalone Sulcotrione

Amidosulfuron Diacetoxyscirpenol (DAS) Fenthion MCPA (-) Pirimicarb T-2 toxin

Asulam Diazinon Fenthion-sulfone Mecoprop (-) Pirimicarb, desmethyl Tebuconazole

Atrazin Dichlorprop (-) Fenthion-sulfoxide Mefenpyr-diethyl Pirimiphos-ethyl Theobromine

Azinphos-methyl Difenconazole Fipronil Mesosulfuron-methyl Pirimiphos-methyl Thiabendazole

Azoxystrobin Difenoxuron Fluazifop Metabenthiazuron Prochloraz Thiacloprid

Bentazon (-) Diflubenzuron Fluazinam (-) Metalaxyl Propamocarb Thiamethoxam

Benzoximate Diflufenican Flufenacet Metamitron Propiconazole Thifensulfuron-methyl

Bifenazate Dimethoate Fluquinconazole Methamidophos Propoxur Thiofanate-methyl

Bromoxynil (-) Dimethomorph-I Fluroxypyr Methiocarb Propyzamid Tolylfluanide

Carbaryl Dimethomorph-II Fomesafen (-) Methiocarb-sulfone Pymetrozin Tralkoxydim

Carbendazim Diuron Fumonisin B1 Methiocarb-sulfoxide Pyrazophos Triadimefon

Carbetamide Dodine Fumonisin B2 Metoxuron Pyridaben Triadimenol

Carbofuran Epoxiconazole Fumonisin B3 Metribuzine Pyridate Triasulfuron

Carbofuran, 3-hydroxy Ergocornine Furathiocarb Monocrotaline Quimerac Triforine-I

Chaconin, alpha Ergocristine Haloxyfop Neburon Retrorsine Triforine-II

Chlorpyriphos Ergocryptine Heliotrine Nicotin Retrorsine-N-oxide Zearalenol, alpha- (-)

Chlorpyriphos-methyl Ergonovine Hexaconazole Ochratoxin-A Senecionine Zearalenol, beta- (-)

Chlorsulfuron Ergotamine Hexythiazox Omethoate Senecionine-N-oxide Zearalenone (-)

Chlortoluron Ethiofencarb HT-2 toxin Oxamyl Seneciphylline

Clofentezine Fenamiphos Imazalil Oxamyl-oxime Seneciphylline, acetyl

Benzimidazole Ionophore Nitroimidazole NSAID Quinolone Sulfonamide

Albendazole Maduramycin Dimetridazole Carprofen Ciprofloxacin Dapson

Albendazole-amidosulfon Monesin HMMNI* Diclofenac Danofloxacin Sulfachloropyridazine

Albendazole-sulfone Narasin Ipronidazole Fenbufen Difloxacin Sulfadiazin

Aminobendazole Salicomicyn Ipronidazole-hydroxide Flufenamic acid Enrofloxacin Sulfadimethoxin

Aminoflubendazole Metronidazole Flunixin Flumequin Sulfadimidine

Fenbendazole Macrolide Metronidazole-hydroxid Indoprofen Marbofloxacin Sulfadoxine

Fenbendazole-sulfone Aivlosin Ronidazole Ketoprofen Nalidixinic Acid Sulfamerazin

Fenbendazole-sulfoxide Erythromycin Ternidazole Meclofenamic acid Norfloxacin Sulfamethizole

Flubendazole Josamycin Naproxen Oxalinic acid Sulfamethoxazole

Levamisole Lincomycin Tranquilizer Niflumic acid Sarafloxacin Sulfamethoxypyridazine

Mebendazole Pirlimycin Azaperon Phenylbutazone Sulfamonomethoxin

Mebendazole, hydroxy Spiramycin Carazolol Piroxycam Tetracycline Sulfamoxole

Oxibendazole Tiamulin Chloropromazine Propyphenazon Chlorotetracycline Sulfapyridin

Thiabendazole Tylosine Haloperidol Tolmetin Doxycyclin Sulfaquinoxalin

Thiabendazole, 5-hydroxy Valnemulin Propionpromazin Oxytetracycline Sulfathiazole

Xylazin Tetracycline Sulfisoxazole

Generic extraction

Luke: aceton + DCM/pentane partitioning

EtAc: ethyl acetate/water partitioning

QuEChERS: acetonitrile/water partitioning (no d-SPE)

Myco: ACN/water 84/16

0

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Luke EtAc Quechers Myco ACN MeOH ACE

% o

f 170 p

esti

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toxin

s/p

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s

<30%

30-50%

50-70%

70-120%

>120%

25% water and 1% FA

Generic extraction: final method

feed

milk

honey

1) take 2.5 g 2) add 10 mL

ACN/H2O/AA

3) shake 30 min 4) Centrifuge

For LC-HRMS:

inject 5 µL

For GC-HRMS:

LLE partitioning (water:ACN)

dSPE (C18/NH2)

inject 5 µL

Instrumental measurement LC & GC with HRMS

LC-ESI-Q-TOF LC-ESI-Q-TOF GC-APCI-Q-TOF

GC-EI-Q-Orbitrap LC-ESI-Q-Orbitrap

GC-EI-Q-TOF

https://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCNjxmc-z7MgCFQp9Ggodaj4ASw&url=https://www.agilent.com/en-us/products/mass-spectrometry/gc-ms-systems/7200-gc-q-tof&psig=AFQjCNGhfboIUV6Dzo06WI8OtvO6iQN5hA&ust=1446370343402479

https://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjZrqH45vPOAhVCvhQKHa5rAZwQjRwIBw&url=https://forschungsinfrastruktur.bmwfw.gv.at/en/fi/massenspectrometer-maxis-impact-q-tof_2743&psig=AFQjCNEMY93VwmX0Pc5oGtT60g_aFyhYyA&ust=1473012918256777

HRMS data acquisition

Needed for selective detection: resolving power of ~50,000 FWHM* Needed for selective screening/identification: At least two ions, preferably including molecular ion/(de)protonated molecule In case hard ionisation (GC-EI): positive ions, 1 injection: 1 scan event = full scan m/z 50-500 (molecular ion, fragments) In case of soft ionisation (ESI, APCI): positive and negative ions, 2 injections: 2 alternating scan events: - full scan m/z 50-1200 w/o fragmentation => (de)protonated molecule and/or adducts - full scan m/z 50-1000 with fragmentation (no precursor ion selection)** => fragment ions

**Vendor terms for fragmentation in a collission cell without precursor ion selection:

(‘All-ion fragmentation’, ‘all ions MS/MS’, ‘MSE’, ‘broad band CID’, .....)

* Kellmann et al, JASMS, 2009, 20, 1464–1476 Mol et al, ACA, 2016, 935, 171-172

Combined Full scan + AIF acquisition

FS: m/z 100-1000 AIF Ϟ m/z 100-1000

Upper & lower m/z cut-off

FS: m/z 100-1000 AIF Ϟ m/z 100-1000

FS: m/z 100-1000 AIF Ϟ m/z 100-1000

Upper & lower m/z cut-off

FS: m/z 100-1000 AIF Ϟ m/z 100-1000

Combined Full scan + AIF acquisition

Analysis result

RT: 0.00 - 20.01

0 2 4 6 8 10 12 14 16 18 20

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1006.68

6.91 7.82 9.88 10.55 12.00 14.82 15.4013.202.23 5.735.213.72 16.98 17.95 18.971.30

7.58

8.02 8.447.35 9.91 10.90 12.12 13.45 15.98 18.58

11.15

6.6811.18

11.92

7.5811.06

15.5014.147.68 12.102.55 9.886.18 15.35 15.822.11 6.08 9.708.475.232.67

16.91 17.290.35 1.29 19.24

NL: 7.00E8

m/z= 195.0868-195.0888 F: FTMS + p ESI Full ms [70.00-1000.00] MS QEx_150618_056

NL: 2.56E8


NL: 1.07E9

TIC F: FTMS + p ESI Full ms [70.00-1000.00] MS QEx_150618_056

Chromatogram Retention time 0-20 min Accurate mass m/z 50-1200 Intensity 6 orders of magnitude

Res

po

nse

→

TIC = total ion current Chromatogram

target analytes hidden in TIC.....

Challenge: finding food toxicants in raw data

Targeted search Dedicated food toxicant database/library - Retention time - 2 diagnostic ions

Search for peaks in the XICs of diagnostic ions at expected retention time + short processing time, fully automated + low LODs ± Scope = number analytes in database - Reference standards needed for tr and selection best 2 ions

Semi-non-target

Non-target

In-house dedicated database (LC-HRMS)

Excel => csv => input tr & diagnostic ions for data handling software

Adducts Isotopes Fragments

Analyte Formula RT (min) [M+H]+ [M+NH4]+ [M+Na]+ [M-H]- 13C 37Cl 81Br 34S Fragment 1 Fragment 1 Fragment 2 Fragment 2

Abamectin B1a I C48H72O14 15.82 0 100 0 0 48 C34H47O7 567.3316 C9H21O11 305.1078

Acetamiprid C10H11ClN4 7.07 100 0 0 10 1 C6H5ClN 126.1050

Acetochlor C14H20ClNO2 11.12 100 10 5 14 1 C10H14N 148.1121 C12H15ClNO 224.0837

Acrinathrin C26H21F6NO5 13.68 0 80 100 26 C14H18NO 208.0757 C13H90 181.0648

Aflatoxin B1 C17H12O6 8.52 100 5 20 17 C16H9O5 281.0444 C16 H11 O6 299.0550

Alachlor C14H20ClNO2 11.15 100 5 1 14 1 C11H16N 162.1277 C13H17ClNO 238.0993

Aldicarb C7H14N2O2S 6.22 0 100 0 0 7 1 x

Aldicarb-sulfone C7H14N2O4S 5.76 50 100 0 0 7 1 C5H10NO2S 148.0427

Aldicarb-sulfoxide C7H14N2O3S 5.52 100 5 5 0 7 1 C4H9S 89.0419

Ametryn C9H17N5S 10.43 100 0 0 9 1 C6H12N5S 186.0808 C4H6N3 96.0556

Amidosulfuron C9H15N5O7S2 8.03 100 0 15 0 9 2 C7H9N4O5S 261.0299

Asulam C8H10N2O4S 5.30 50 100 50 0 8 1 C6H6O2NS 156.0114

.......

Bentazone C10H12N2O3S 7.09 0 0 0 100 10 1 C7H4ON2 132.0329

Bromoxynil C7H3Br2NO 8.62 0 0 0 100 7 2 Br[81] 80.9168 Br[79] 78.9189

.......

Carbetamide C12H16N2O3 8.44 25 0 100 100 12 C5H12NO2 118.0863 C7H6NO 120.0444

Chlorantraniliprole C18H14N5O2BrCl2 9.85 100 0 50 100 18 1 2 C14H4BrCl 285.9179

.......

Chromafenozide C24H30N2O3 10.80 100 0 10 100 24 C11H11O2 175.0754

Clothianidin C6H8ClN5O2S 6.91 100 1 8 100 6 1 1 C4H3NClS 131.9669

Cyclanilide C11H9Cl2NO3 9.41 1 100 0 100 11 2 C6H3NCl 123.9949

.......

Deoxynivalenol C15H20O6 6.02 75 25 100 15 C13H15O2 203.1067 C13H14O4 234.0887

.......

Propoxur C11H15NO3 8.74 100 1 1 0 11 C6H7O2 111.0441 C6H5O 93.0335

.......

Spinosyn A C41H65NO10 11.93 100 0 1 0 41 C7H16NO 142.1226 C6H12N 98.0964

Sulcotrione C14H13ClO5S 7.02 50 100 20 100 14 1 1 C14H13O5S 293.0478

.......

XMC C10H13NO2 9.24 100 10 5 0 10 C8H11O 123.0804

RT: 0.00 - 25.02 SM: 7B

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1007.65

11.35

7.40

8.54 8.57

8.44

5.58

5.99

5.56

NL: 5.20E4

m/z= 192.07574-192.07766 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29

NL: 1.30E5


NL: 6.28E4


NL: 1.29E4


NL: 9.93E4


NL: 6.08E4


RT: 0.00 - 25.02 SM: 7B

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1.92

19.64

19.38

19.11

19.05 21.43 21.70

21.8718.96

22.01

16.36

16.15

13.44

11.55

NL:3.76E8

TIC F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29

m/z 308.1524 C16H22ClN3O

RT: 0.00 - 25.02 SM: 7B

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1007.65

11.35

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8.54 8.57

8.44

5.58

5.99

5.56

NL: 5.20E4


NL: 1.30E5


NL: 6.28E4


NL: 1.29E4


NL: 9.93E4


NL: 6.08E4


tebuconazole

Honey spiked with pesticides, veterinary drugs, plant toxins at 10 µg/kg

m/z 311.0809 C12H14N4O4S

RT: 0.00 - 25.02 SM: 7B

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1007.65

11.35

7.40

8.54 8.57

8.44

5.58

5.99

5.56

NL: 5.20E4


NL: 1.30E5


NL: 6.28E4


NL: 1.29E4


NL: 9.93E4


NL: 6.08E4


sulfadimethoxine

RT: 0.00 - 25.02 SM: 7B

0 5 10 15 20 25

Time (min)

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1007.65

11.35

7.40

8.54 8.57

8.44

5.58

5.99

5.56

NL: 5.20E4


NL: 1.30E5


NL: 6.28E4


NL: 1.29E4


NL: 9.93E4


NL: 6.08E4


m/z 916.5264 C46H77NO17

tylosine

RT: 0.00 - 25.02 SM: 7B

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1007.65

11.35

7.40

8.54 8.57

8.44

5.58

5.99

5.56

NL: 5.20E4


NL: 1.30E5


NL: 6.28E4


NL: 1.29E4


NL: 9.93E4


NL: 6.08E4


m/z 290.1751 C17H23NO3

atropine

RT: 0.00 - 25.02 SM: 7B

0 5 10 15 20 25

Time (min)

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1007.65

11.35

7.40

8.54 8.57

8.44

5.58

5.99

5.56

NL: 5.20E4


NL: 1.30E5


NL: 6.28E4


NL: 1.29E4


NL: 9.93E4


NL: 6.08E4


m/z 336.1805 C18H25NO5

senecionine

Extracting toxicants from the raw data m/z 192.0767 C9H9N3O2

RT: 0.00 - 25.02 SM: 7B

0 5 10 15 20 25

Time (min)

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1007.65

11.35

7.40

8.54 8.57

8.44

5.58

5.99

5.56

NL: 5.20E4


NL: 1.30E5


NL: 6.28E4


NL: 1.29E4


NL: 9.93E4


NL: 6.08E4


carbendazim

Extract signal of exact mass ± x Da (ppm)

e.g. [M+H]+ 336.1805 ± 0.002 Da (± 6 ppm)

Example need for 2nd ion for more selective

detection/identification

Simazine [M+H]+ 202.0854

Carbaryl [M+H]+ 202.0863

Simazine C7H12ClN5 and Carbaryl C12H11NO2 : difference [M+H]+ = 0.9 mDa (4 ppm)

Carbaryl Fragment 145.0648

Simazine Fragment 124.0869

Spiked Leek FS 70K, vDIA 35K

RT: 8.15 - 10.15 SM: 5G

8.5 9.0 9.5 10.0

Time (min)

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Re

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Ab

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1009.13 9.16

9.11

9.17

9.18

NL: 1.81E7


NL: 7.50E5

m/z= 124.08630-124.08754 F: FTMS + p ESI Full ms2 [email protected] [50.00-225.00] MS QEx_140918_102

NL: 1.60E7


NL: 3.09E7


XICs MEW ±5 ppm

Full

scan

wit

h

frag

men

tati

on

Fu

ll sc

an w

/o

frag

men

tati

on

RT: 8.15 - 10.15 SM: 5G

8.5 9.0 9.5 10.0

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Ab

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9.11

9.17

9.18

NL: 1.81E7


NL: 7.50E5


NL: 1.60E7


NL: 3.09E7


2 ions boscalid

List with pesticides automatically found by screening software

Screening: data review

Challenges of targeted screening 1000+ analytes

2. HRMS database / library

- In-house = optimal but time intensive

- Vendor = inflexible (fixed instrument/conditions)

- Generic (MS only, no tr)

EI-MS: instrument independent NIST library: >275,000 spectra, but not high res HRMS libraries in development

ESI/APCI-MS: instrument/setting dependent

HRMS libraries acquired with multiple instruments/conditions in development

1. Fit-for-purpose/user friendly data processing software

- Vendor software

- Generic, e.g. MetAlign http://www.wur.nl/en/show/MetAlign-1.htm

http://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCN7e7pqY78gCFcjXGgod6lwKxQ&url=http://www.gcimage.com/nistlib.html&psig=AFQjCNHHgnbh83dyUJ0g-Fcu_TxdJY1VEQ&ust=1446466028830469

https://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiSkJLOtvfMAhXE0hoKHcm4CvUQjRwIBw&url=https://www.pinterest.com/pin/129548926756059638/&bvm=bv.122852650,d.d2s&psig=AFQjCNE_FqzHvL_v0LXh6SbiDm6vmW2TrQ&ust=1464341301851045

http://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=&url=http://www.moneysavingexpert.com/news/phones/2015/05/that-smile-might-cost-you-beware-being-hit-with-huge-emoji-fees&psig=AFQjCNFX1f99z0sw2O8fRpd3GUUAgclGug&ust=1464406450367083

http://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjC9KeptvfMAhUL6xoKHTTWAN0QjRwIBw&url=http://www.redbubble.com/people/meje/works/19204822-worried-face-emoji?p=throw-pillow&bvm=bv.122852650,d.d2s&psig=AFQjCNEAyEl7BxsI3dk6vukFjs8bRw9nCg&ust=1464341225196501





http://www.wur.nl/en/show/MetAlign-1.htm




https://www.mzcloud.org/

Example generic libraries for LC-HRMS

Challenges of targeted screening 1000+ analytes

3. Optimisation of peak detection parameters

4. Validation / assessment reliability / LODs

Multiple parameters..... Settings determine rate of false negatives and false positives

Peak detection algorithms Filtering: ∆ tr ∆ mass accuracy Isotope fits Fragments Spectral fits Response thresholds ........

Count # pesticides found in each sample detection rate / false negatives blank samples: false positives

Validation set: 11 matrices: Non-fortified, 3 fortifications: 0.01, 0.05 and 0.20 mg/kg Test set for fortification: 183 pesticides Analysed on 2 different days (4 weeks in between) => 22 samples (LC-HRMS)

Example from pesticides in the EU: Guidance document SANTE/11945/2015*

Determine detection rate for target compound in ≥20 samples at a target concentration Reliability is considered acceptable when detection rate ≥95% (false negative <5%)

Reliability of automated screening

http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_guidelines_wrkdoc_11945_en.pdf

*





Reliability of automated screening

0

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10 50 200 ??

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es t

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Fortification level (µg/kg)

Method performance per analyte

0

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100 Apple

Necatarine

Leek

Tomato

Broccoli

Lettuce Celery

Beans

Carrot

Orange

Grapes

200 ppb 50 ppb 10 ppb

Detection rates various matrices as % of spiked pesticides / sample:

Overall detections in spiked samples (4026 pesticide/matrix combinations per level): 0.01 mg/kg: 91.9% 0.05 mg/kg: 97.2% 0.20 mg/kg: 98.3%

Finding food toxicants in raw data

Targeted search Dedicated MS features or scripts - Cl, Br clusters - Common fragments

Search for MS features in entire chromatogram + Can detect unknowns - Only detects compounds containing the feature ± LODs higher than targeted search - Medium/long processing time, more difficult to automate - Identification needs further investigation and confirmation against reference standard

Semi-non-target

Non-target

Screening for designer drugs/structural analogues Viagra (sildenafil, PDE-5 inhibitors): more than 80 designer analogues known

sildenafil

Adulterants: change O by S

change group

change link

Example semi non-target detection

Analogues => MS feature = common fragments


C5H11N2 = 99.09167 C5H12N2 = 100.09950

sildenafil

Common fragments in ESI+ for many analogues

Common fragments in ESI- for many analogues

ESI+

C15H14O2N4 282.11222

ESI-

S

S-analogue

S

C15H14ON4S 298.08938

Detection of analogues through common fragments


RT: 2.00 - 16.00 SM: 3B

2 4 6 8 10 12 14 16

Time (min)

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10011.73

11.78

11.13

9.87 11.916.04

12.096.17 15.522.55 9.68

11.73

11.78

11.13

9.87 11.916.04

12.096.17 15.522.55 9.68

10.05

11.73

NL: 1.30E9

TIC F: FTMS + p ESI Full ms [70.00-1000.00] MS QEx_150618_051_374566_100x

NL: 1.30E9

TIC F: FTMS + p ESI Full ms [70.00-1000.00] MS QEx_150618_051_374566_100x

NL: 1.34E7

m/z= 475.20982-475.21458 F: FTMS + p ESI Full ms [70.00-1000.00] MS QEx_150618_051_374566_100x

NL: 3.32E7

m/z= 99.09117-99.09217 F: FTMS + p ESI Full ms2 [email protected] [50.00-535.00] MS QEx_150618_051_374566_100x

Sildenafil identified, but far below therapeutic level 0.68 μg/g; 0.4 mg/dose

RT: 2.00 - 16.00 SM: 3B

2 4 6 8 10 12 14 16

Time (min)

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6.05

2.01 12.152.57 6.19 8.195.34 14.9510.03

10.03

10.03

10.83

11.74

11.91

NL: 5.25E8

TIC F: FTMS - p ESI Full ms [75.00-1000.00] MS QEx_150618_017_374566_100x_neg

NL: 1.00E7

m/z= 473.19528-473.20002 F: FTMS - p ESI Full ms [75.00-1000.00] MS QEx_150618_017_374566_100x_neg

NL: 5.38E5

m/z= 282.11081-282.11363 F: FTMS - p ESI Full ms2 [email protected] [50.00-535.00] MS QEx_150618_017_374566_100x_neg

NL: 2.34E7

m/z= 298.08789-298.09087 F: FTMS - p ESI Full ms2 [email protected] [50.00-535.00] MS QEx_150618_017_374566_100x_neg

ESI+ ESI- TIC TIC

Common fragment C5H11N2 99.09167

Common fragment C15H14O2N4 282.11222

Sildenafil M+H 475.21220

Sildenafil M-H 473.19765

Common fragment S-analogues C15H14ON4S 298.08938

OK

OK

OK

OK

?

?

?

Frag

men

tati

on

sca

n

QEx_150618_051_374566_100x #4195-4208 RT: 11.75-11.78T: FTMS + p ESI Full ms [70.00-1000.00]

491 492 493

m/z

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491.18974

492.19316

493.18374

Full

scan

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men

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Fu

ll sc

an w

/o

frag

men

tati

on

QEx_150618_051_374566_100x #4195-4208 RT: 11.75-11.78T: FTMS + p ESI Full ms [70.00-1000.00]

491 492 493

m/z

0

10

20

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Re

lative

Ab

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491.18974

492.19316

493.18374

Accurate masses + isotopic pattern: Limits the options for molecular formula

C22H22N10O4 491.18983

C29H28ClFN2O2 491.18961

C30H26N4OS 491.19001

C22H30N6O3S2 491.18936

Identification of the ‘unknown’



Manual evaluation of the options.....

Structural analogue of sildenafil


=> Tentatively identified as thiosildenafil Final confirmation by analysis against reference standard

Structural analogue of sildenafil Compound a.k.a. thiosildenafil

Finding food toxicants in raw data

Targeted search

Pre-processing/deconvolution/peak picking of entire chromatogram/raw data Data w/o fragmentation: accurate mass isotopic pattern 7 Golden rules*

Semi-non-target

Non-target

*Kind & Fiehn, BMC Bioinformatics (2007) 8: 105

Data with fragmentation (e.g. GC-EI-HRMS):

mol. formula

MS spectra

+ Scope is 105-106 compounds + Can detect unknowns - Long processing time, - Very high number of hits, most not

meaningful/not of interest - Identification needs lots of further

investigation and finally confirmation against ref. standard

Search spectral libraries

Search compound databases: PubChem, ChemSpider

RT: 0.00 - 20.01

0 2 4 6 8 10 12 14 16 18 20

Time (min)

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100

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1006.68

6.91 7.82 9.88 10.55 12.00 14.82 15.4013.202.23 5.735.213.72 16.98 17.95 18.971.30

7.58

8.02 8.447.35 9.91 10.90 12.12 13.45 15.98 18.58

11.15

6.6811.18

11.92

7.5811.06

15.5014.147.68 12.102.55 9.886.18 15.35 15.822.11 6.08 9.708.475.232.67

16.91 17.290.35 1.29 19.24

NL: 7.00E8


NL: 2.56E8


NL: 1.07E9

TIC F: FTMS + p ESI Full ms [70.00-1000.00] MS QEx_150618_056

Issue: often 1000s unique peaks in one sample, too many for searching against compound data bases.... But might work when restricting to highest peaks (high concentrations)

Non-target detection

Investigate ions for the highest peaks in the TIC

Example: food supplement screening

RIK no File Retention Exp. M0 Intensity Delta mass Other delta masses Num. isotope massesFormula Theor. M0 ppm Mass Analysis Report CSV database Chemspider link Num. Pubchem entriesComment

316415 Exa_131101_060_recal 6.36 299.1753 9,343,537 -1.007276 H; 2 C18H22N2O2 298.168128 -0.270091 1 solutions (R)-pyridin-4-yl[4-(2-pyrrolidin-1-ylethoxy)phenyl]methanol; (2S)-1-(9H-Carbazol-4-yloxy)-3-(isopropylamino)propan-2-olC18H22N2O2() 5574

316415 Exa_131101_060_recal 1.82 160.0968 10,404,089 -1.007276 K;Na;H; 3 C7H13N1O3 159.089543 0.081265 1 solutions 1-Amino-2,3-Dihydroxy-5-Hydroxymethyl Cyclohex-5-Ene C7H13N1O3() 1634

316415 Exa_131101_060_recal 10.60 343.1177 21,494,452 -1.007276 H; 3 C19H18O6 342.110338 0.179511 2 solutions 2-Benzo[1,3]Dioxol-5-Ylmethyl-3-Benzyl-Succinic Acid C19H18O6() 844

316415 Exa_131101_060_recal 10.51 365.0995 5,888,131 -22.989216 K;Na; 3 C19H18O6 342.110338 -0.017016 2 solutions 2-Benzo[1,3]Dioxol-5-Ylmethyl-3-Benzyl-Succinic Acid C19H18O6() 844

316415 Exa_131101_060_recal 8.65 207.0652 11,685,853 -1.007276 K;Na;H; 3 C11H10O4 206.057909 -0.070754 1 solutions 4-(2-METHOXYPHENYL)-2-OXOBUT-3-ENOIC ACID C11H10O4() 953

316415 Exa_131101_060_recal 6.38 195.0876 486,248,352 -1.007276 Na;H; 1 C8H10N4O2 194.080376 -0.183828 1 solutions Caffeine; Enprofylline C8H10N4O2() 663 SATURATION

316415 Exa_131101_060_recal 7.22 195.0876 39,819,664 -1.007276 H;H-2H2O; 3 C8H10N4O2 194.080376 -0.105207 1 solutions Caffeine; Enprofylline C8H10N4O2() 663

316415 Exa_131101_060_recal 10.71 195.0876 29,205,226 -1.007276 H; 3 C8H10N4O2 194.080376 -0.105207 1 solutions Caffeine; Enprofylline C8H10N4O2() 663

316415 Exa_131101_060_recal 10.86 195.0877 20,606,294 -1.007276 H; 3 C8H10N4O2 194.080376 0.130656 1 solutions Caffeine; Enprofylline C8H10N4O2() 663

316415 Exa_131101_060_recal 11.37 195.0877 12,032,150 -1.007276 H; 1 C8H10N4O2 194.080376 0.052035 1 solutions Caffeine; Enprofylline C8H10N4O2() 663

316415 Exa_131101_060_recal 2.49 195.0877 10,449,775 -1.007276 NH4;H; 3 C8H10N4O2 194.080376 0.287898 1 solutions Caffeine; Enprofylline C8H10N4O2() 663

316415 Exa_131101_060_recal 4.87 150.0913 6,151,225 -1.007276 H;H-H2O; 2 C9H11N1O1 149.084064 -0.00622 1 solutions Cathinone C9H11N1O1() 1369

316415 Exa_131101_060_recal 5.82 289.0707 25,799,604 17.003284 H;H-H2O; 4 C15H14O7 306.073953 0.233265 1 solutions Epigallocatechin C15H14O7() 145

316415 Exa_131101_060_recal 5.58 307.0813 18,737,092 -1.007276 K;Na;H;H-H2O; 3 C15H14O7 306.073953 0.228591 2 solutions Epigallocatechin C15H14O7() 145

316415 Exa_131101_060_recal 7.40 303.0863 13,134,170 -1.007276 H; 3 C16H14O6 302.079038 0.066169 2 solutions Hesperetin C16H14O6() 580

316415 Exa_131101_060_recal 11.03 290.1752 13,712,555 -1.007276 K;Na;H; 3 C17H23N1O3 289.167794 0.455611 1 solutions Hyoscyamine; Atropine C17H23N1O3() 6115

316415 Exa_131101_060_recal 6.36 138.0662 21,984,322 -1.007276 H; 2 C6H7N3O1 137.058912 -0.188322 1 solutions Isoniazid C6H7N3O1() 429

316415 Exa_131101_060_recal 5.35 166.0862 220,955,152 -1.007276 K;Na;H; 3 C9H11N1O2 165.078979 -0.257468 1 solutions L-Phenylalanine; Benzocaine; D-Phenylalanine; 4-Aminohydrocinnamic Acid; Tyrosinal; 4-(DIMETHYLAMINO)BENZOIC ACID; 2-(4-METHOXYPHENYL)ACETAMIDEC9H11N1O2() 2146

316415 Exa_131101_060_recal 5.13 166.0862 166,244,768 -1.007276 K;Na;H; 3 C9H11N1O2 165.078979 -0.257468 1 solutions L-Phenylalanine; Benzocaine; D-Phenylalanine; 4-Aminohydrocinnamic Acid; Tyrosinal; 4-(DIMETHYLAMINO)BENZOIC ACID; 2-(4-METHOXYPHENYL)ACETAMIDEC9H11N1O2() 2146

316415 Exa_131101_060_recal 1.94 116.0706 9,224,306 -1.007276 K;H;H-H2O;H-2H2O; 2 C5H9N1O2 115.063329 -0.21786 1 solutions L-Proline; D-Proline; N-Methyldehydrobutyrine C5H9N1O2() 800

316415 Exa_131101_060_recal 1.79 118.0863 6,271,322 -1.007276 K;Na;H; 3 C5H11N1O2 117.078979 0.093127 1 solutions L-Valine; Amyl Nitrite; N,N-Dimethyl-L-Alanine; 2-Amino-Pentanoic Acid; D-Isovaline; NorvalineC5H11N1O2() 676

316415 Exa_131101_060_recal 1.69 104.107 19,628,580 -1.007276 H; 3 C5H13N1O1 103.099714 -0.1041 1 solutions L-Valinol C5H13N1O1() 244

316415 Exa_131101_060_recal 10.79 286.1438 447,307,328 -1.007276 Na;H; 4 C17H19N1O3 285.136493 -0.00431 1 solutions Morphine; Hydromorphone C17H19N1O3() 6970

316415 Exa_131101_060_recal 7.31 273.0757 65,387,364 -1.007276 H; 4 C15H12O5 272.068473 -0.018102 2 solutions Naringenin C15H12O5() 694

316415 Exa_131101_060_recal 6.39 273.0756 5,182,375 -1.007276 H; 2 C15H12O5 272.068473 -0.466777 2 solutions Naringenin C15H12O5() 694

316415 Exa_131101_060_recal 6.71 229.0859 7,135,361 -1.007276 H; 2 C14H12O3 228.078644 0.07537 1 solutions Oxybenzone; Resveratrol; Trioxsalen C14H12O3() 1189

316415 Exa_131101_060_recal 7.69 229.0859 6,607,085 -1.007276 Na;H; 3 C14H12O3 228.078644 0.07537 1 solutions Oxybenzone; Resveratrol; Trioxsalen C14H12O3() 1189

316415 Exa_131101_060_recal 2.55 150.0913 84,124,608 17.003284 Na;H;H-H2O; 3 C9H13N1O2 167.094629 -0.213164 1 solutions Phenylephrine; Metaraminol; Ethinamate; PHENYLALANINDIOLC9H13N1O2() 2311

316415 Exa_131101_060_recal 3.28 150.0914 5,218,466 17.003284 H;H-H2O; 2 C9H13N1O2 167.094629 0.152109 1 solutions Phenylephrine; Metaraminol; Ethinamate; PHENYLALANINDIOLC9H13N1O2() 2311

316415 Exa_131101_060_recal 4.70 152.107 13,409,422 -1.007276 NH4;H;H-H2O; 3 C9H13N1O1 151.099714 -0.172015 1 solutions Phenylpropanolamine; Cathine; L-Phenylalaninol C9H13N1O1() 1812

316415 Exa_131101_060_recal 4.37 152.107 11,333,827 -1.007276 H; 2 C9H13N1O1 151.099714 -0.07103 1 solutions Phenylpropanolamine; Cathine; L-Phenylalaninol C9H13N1O1() 1812

316415 Exa_131101_060_recal 8.80 303.0498 40,617,912 -1.007276 K;Na;H; 4 C15H10O7 302.042653 -0.309654 1 solutions Quercetin; 5,7-DIHYDROXY-2-(3,4,5-TRIHYDROXYPHENYL)-4H-CHROMEN-4-ONEC15H10O7() 109

316415 Exa_131101_060_recal 7.48 633.1426 8,365,348 -22.989216 K;Na;H; 4 C27H30O16 610.153385 -0.037109 32 solutions Rutin C27H30O16() 232

316415 Exa_131101_060_recal 1.99 130.0862 5,135,142 -1.007276 K;Na;H; 2 C6H11N1O2 129.078979 -0.447488 1 solutions Vigabatrin; (4e)-4-Aminohex-4-Enoic Acid; Cycloleucine C6H11N1O2() 1581

Screen for adulteration with pharmaceuticals Data pre/post processing: MetAlign/Extract_LC Databases: drugbank (5000 substances) & Chemspider

http://www.drugbank.ca http://www.wageningenur.nl/en/show/MetAlign-1.htm

http://www.drugbank.ca/

http://www.wageningenur.nl/en/show/MetAlign-1.htm



http://www.google.nl/url?sa=i&source=images&cd=&cad=rja&uact=8&docid=820ZYHtwUlDCLM&tbnid=wI6M_3AQub6E3M&ved=0CAgQjRw&url=http://www.iconarchive.com/show/swanky-outlines-icons-by-pixelkit/00-Pointer-Hand-icon.html&ei=te4lU9awAaPD0QXJw4HIDw&psig=AFQjCNFNnXKykahJlSO46yYG0TKPMouGXA&ust=1395081269126021

Identification of C9H13NO

Narrowing down the # options

Use availabe meta-information: e.g. known or claimed bio-effects

2) MS database search for known β-agonist ‘effect directed database searching’

Weightloss / stimulant health complaints by users (nausea, tachycardia, 1x cardiac arrest)

β-agonist?

Dexaprine case, Netherlands, 2013

1)Bioassay: β–adrenoceptor competitive binding assay: positive!

Isopropyloctopamine is a known beta agonist....

With the right database and meta-information many ‘unknowns’ become knowns and easy/easier targets!

Isopropyloctopamine found at high level (20 g/kg ≡ 60 mg/dose) Note: not in drugbank!

Narrowing down the # options

https://www.google.nl/url?q=http://www.dexaprine.org/ingredients/isopropyloctopamine&sa=U&ei=MvSVUuC7LMWyywPus4HIBA&ved=0CCoQ9QEwAA&usg=AFQjCNF2zIBehbZbTPU-nWpLFcXKZD5NsQ

- Scope compromises LOD, for LODs <<10 μg/kg) more dedicated methods involving more cleanup are required

- e.g. dioxins, growth promotors/steroids

- Not all toxicants are amenable to generic methods - e.g. extremely polar, extremely volatile, instability, ...

- Not high-throughput

Limitations of wide scope screening methods

Chemical screening

No time to waste Screening in seconds...

Applications: Detection of specific toxicants not amenable to generic methods Fast screening in case of intoxications / adulteration

Rapid chemical screening methods

Eliminate the time-limiting step = chromatography

Flow injection – mass spectrometry

FI-ESI-MS

Ambient MS

e.g. DART-MS

Flow injection – MS/MS

autosampler

injector

HPLC

column

eluent

MS/MS detector

No chromatographic problems Eluent optimal for MS sensitivity (no compromise with LC)

Fast: 5-20 seconds

No additional instrument requirements (↔ ambient MS sources)

Example application: highly polar pesticides

Pesticide Precursor Transition-1 Transition-2

Amitrole [M+H]+85 > 58 85 > 43

Chlormequat [M]+122 > 58 122 > 63

Cyromazine [M+H]+167 > 108 167 > 125

Daminozide [M+H]+161 > 143 161 > 61

Diquat [M]+184 > 128 184 > 158

Mepiquat [M]+114 > 98 114 > 70

Paraquat [M]+186 > 155 186 > 77

AMPA [M-H]-110 > 63 110 > 79

Ethephon [M-H]-143 > 79 143 > 107

Fosetyl-Al [M-H]-109 > 63 109 > 81

Glufosinate [M-H]-180 > 63 180 > 85

Glyphosate [M-H]-168 > 63 168 > 81

Maleic hydrazide [M-H]-111 > 82 -

Sample preparation

10 g fruit/veg 2.5 g dry cereals

30 min end-over-end 10 mL water

centrifuge

raw extract (0.25-0.5 g/mL)

10x dilution of raw extract with optimum ‘eluent’ in filter vial

Matrix effects

Response relative to solvent standard (%)

Example applications: Chlormequat

Oats with incurred chlormequat Assigned value 0.479 mg/kg

measured

1 0.466

2 0.471

3 0.476

4 0.488

5 0.489

average (mg/kg) 0.478

RSD (%) 2.2

rela

tive

res

po

nse

ng/mL solvent

0.05

mg/kg

blank

Grapes (MRL = 0.05* mg/kg)

blank

0.05

mg/kg

Milk (MRL = 0.05* mg/kg)

H.G.J. Mol, R.C.J. van Dam. Anal Bioanal Chem (2014) 406:6817–6825.

Example applications: Glyphosate

EUPT C5/SRM6 (2011) Rice spiked with Glyphomax Assigned value 0.294 mg/kg

measured

1 0.397

2 0.329

3 0.340

4 0.357

5 0.387

average (mg/kg) 0.362

RSD (%) 8.1

swine kidney MRL = 0.5 mg/kg

0.20

mg/kg

blank

1.0

mg/kg

H.G.J. Mol, R.C.J. van Dam. Anal Bioanal Chem (2014) 406:6817–6825.

Rapid detection intoxication cases: FIA-HRMS

Japanese ← Star anise → Chinese Althaea officinalis ↔ Atropa belladonna

Herbal tea

D:\USERDATA\...\Exa_130322_088 22/03/2013 16:55:10 steranijs 10^4

RT: 0.00 - 2.09

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

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1.261.03

NL: 1.76E5

m/z= 327.10686-327.11014 F: FTMS {1,3} - p ESI Full ms [60.00-1000.00] MS Exa_130322_088

Exa_130322_088 #20-46 RT: 0.40-0.73 AV: 6 NL: 1.45E5T: FTMS {1,3} - p ESI Full ms [60.00-1000.00]

316 318 320 322 324 326 328 330 332 334 336 338 340 342 344

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

327.10864

343.10365328.11239322.91401 324.99492316.94786 333.11166 336.88783 339.19998318.97582 344.97923330.97941

XIC anisatine 327.10745

ESI-

+3.3 ppm

D:\USERDATA\...\Exa_130322_090 22/03/2013 17:00:58 Thee 10^4

RT: 0.00 - 2.10

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

0.50

0.71

1.17 1.64 1.95

NL: 1.34E7


Exa_130322_090 #18-51 RT: 0.37-0.85 AV: 8 NL: 1.41E7T: FTMS {1,1} + p ESI Full ms [60.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

338.34131

290.17456

125.98614

141.95841 365.10502228.19558

407.25390 512.32372 675.67035578.35958 737.49462 825.54810 913.60037

XIC atropine 290.17507

-1.8 ppm

//upload.wikimedia.org/wikipedia/commons/3/3d/Anisatin.PNG

//upload.wikimedia.org/wikipedia/commons/6/64/Atropine.svg

Food fraud: melamine as ‘protein’ adulterant

EU 594/2012: Food 2.5 mg/kg, Powdered infant/follow-on formulae 1 mg/kg EU 107/2013: Feed 2.5 mg/kg

2.5 g milk powder + 13C315N3-melamine

Reconstitute in water Centrifuge 100 µL + 900 µL 0.1%NH3/ACN Filter vial FI-ESI-MS/MS (API5500)

Ion ratios all within ±20% Blank 2.5 mg/kg

rec 87% rsd 0.8%

Soybean meal

m/z 127>68

m/z 127>85

Blank 1 mg/kg 25 ng/mL

rec 90% rsd 3.2%

Infant formulae Solvent std

//upload.wikimedia.org/wikipedia/commons/0/05/Melamine.svg

Food fraud: unknown ‘protein’ adulterants

D:\USERDATA\...\Exa_130610_050 10/06/2013 18:14:31 Milkpowder + 1000 ppm

RT: 0.00 - 2.02

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

0.450.48

0.38

0.280.25

0.670.15 0.79 0.970.92 1.231.19 1.26 1.45 1.761.50 1.911.630.08

NL:1.58E8

TIC MS Exa_130610_050


60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

104.10679

163.05932C 4 H 9 O 4 N 3

180.08589C 4 H 12 O 4 N 4145.04892 198.09654

C 5 H 10 O N 8

127.0386285.0285399.04410 116.98574

105.11012

81.0336969.03381 149.0115991.03903

191.03813C H 9 O 8 N 3168.10650144.06497

Milk powder extract 0.01 g/mL FI-ESI-full scan HRMS

D:\USERDATA\...\Exa_130610_050 10/06/2013 18:14:31 Milkpowder + 1000 ppm

RT: 0.00 - 2.02

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

0.450.48

0.38

0.280.25

0.670.15 0.79 0.970.92 1.231.19 1.26 1.45 1.761.50 1.911.630.08

NL:1.58E8

TIC MS Exa_130610_050


127.00 127.05 127.10 127.15 127.20 127.25 127.30 127.35

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

127.03862 127.07224C 3 H 7 N 6

TIC milk powder extract

Food fraud: unknown ‘protein’ adulterants

exact mass

elemental

composition

113.0589 C4H7ON3

126.0654 C3H6N6

131.0695 C4H9O2N3

133.0276 C6H3ON3

138.0429 C6H6O2N2

141.0273 CHON8

146.1055 C6H14O2N2

155.0695 C6H9O2N3

156.0396 C3H4O2N6

158.9916 C3HO5N3

163.0593 C4H8O4N3

173.0913 C5H11O2N5

175.0957 C6H13O3N3

176.0685 C6H6N7

178.0477 C5H4ON7

179.0780 C4H11O4N4

181.0263 C8HN6

182.0426 C4H4O2N7

184.0345 C4H4O3N6

190.0311 CH8O8N3

197.0899 C5H9ON8

203.0794 C7H7N8

208.0835 C8H10NO2N4

211.0467 C7H7O4N4

218.0188 C7H2O3N6

220.0179 CH2O5N9

exact mass

elemental

composition

% (w/w)

N/compound

141.0273 CHON8 79%

126.0654 C3H6N6 67%

218.0188 C7H2O3N6 58%

220.0179 CH2O5N9 57%

197.0899 C5H9ON8 57%

176.0685 C6H6N7 56%

203.0794 C7H7N8 55%

178.0477 C5H4ON7 55%

182.0426 C4H4O2N7 54%

156.0396 C3H4O2N6 54%

181.0263 C8HN6 46%

184.0345 C4H4O3N6 46%

173.0913 C5H11O2N5 40%

113.0589 C4H7ON3 37%

131.0695 C4H9O2N3 32%

133.0276 C6H3ON3 32%

179.0780 C4H11O4N4 31%

155.0695 C6H9O2N3 27%

208.0835 C8H10NO2N4 27%

211.0467 C7H7O4N4 27%

158.9916 C3HO5N3 26%

163.0593 C4H8O4N3 26%

175.0957 C6H13O3N3 24%

190.0311 CH8O8N3 22%

138.0429 C6H6O2N2 20%

146.1055 C6H14O2N2 19%

exact mass

elemental

composition

% (w/w)

N/compound

hits in

pubchem including

141.0273 CHON8 79% 0

126.0654 C3H6N6 67% 26 melamine

218.0188 C7H2O3N6 58% 0

220.0179 CH2O5N9 57% 0

197.0899 C5H9ON8 57% 0

176.0685 C6H6N7 56% 1

203.0794 C7H7N8 55% 1

178.0477 C5H4ON7 55% 2

182.0426 C4H4O2N7 54% 0

156.0396 C3H4O2N6 54% 9

181.0263 C8HN6 46% 0

184.0345 C4H4O3N6 46% 16

173.0913 C5H11O2N5 40% 33

113.0589 C4H7ON3 37% 340 creatinine

131.0695 C4H9O2N3 32% 180 creatine

133.0276 C6H3ON3 32% 44

179.0780 C4H11O4N4 31% 0

155.0695 C6H9O2N3 27% 1059 histidine

208.0835 C8H10NO2N4 27% 29

211.0467 C7H7O4N4 27% 8

158.9916 C3HO5N3 26% 2

163.0593 C4H8O4N3 26% 1

175.0957 C6H13O3N3 24% 207 citrulline

190.0311 CH8O8N3 22% 0

138.0429 C6H6O2N2 20% 487

146.1055 C6H14O2N2 19% 1036 lysine

‘Agriforensics’

Organic or not ? Non-systemic pesticides, e.g. post-harvest treatment with fungicides

Swap sampling: Spray surface with org. solv.*

*0.1% FA in MeOH/IPA/Ace/Hex/EtOAc Edison et al, Rapid Commun. Mass Spectrom. (2011) 25:127-139

Food ‘forensics’

Swap sampling: Spray surface with org. solv.* Swap with foam Desorb with 2 mL MeOH FI-ESI-HRMS

*0.1% FA in MeOH/IPA/Ace/Hex/EtOAc Edison et al, Rapid Commun. Mass Spectrom. (2011) 25:127-139

Organic or not ? Non-systemic pesticides, e.g. post-harvest treatment with fungicides

RT: 0.00 - 0.80

0.0 0.2 0.4 0.6 0.8

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0.35 0.40

0.43

0.21

0.57

0.64

0.09

0.32

0.24

0.35

0.43

0.570.720.09

NL:1.30E6

m/z= 202.04229-202.04431 MS Exa_130610_148

NL:2.90E6

m/z= 297.05401-297.05699 MS Exa_130610_148

RT: 0.00 - 0.80

0.0 0.2 0.4 0.6 0.8

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

NL:2.20E7

m/z= 202.04229-202.04431 MS Exa_130610_102

NL:4.66E6

m/z= 297.05401-297.05699 MS Exa_130610_102

imazalil

thiabendazole

Organic blank

100 ppb spike ≡ 25 µg

2.6E+07

2.2E+07

‘Agriforensics’

Use of non-registered pesticides?

trunk swap

glass swap

nozzle swap

crop swap

ng amounts easily detected

Conclusions

Possibilities for chemical screening for food/feed toxicants have greatly improved in the past years, mainly due advances in full scan HRMS technologies

Current methods allow automated detection of 1000+ analytes in one run

Dedicated target databases are most effective for screening

Alternative data processing enables detection of (un)knowns beyond the target databases

Flow injection-MS/MS or –HRMS is a useful option for rapid detection of toxicants not amenable to generic screening methods or for situations where speed/high throughput is required

Thank you for your attention!

感谢您的关注！

Many thanks to the many RIKILT colleagues

contributing to this work

Taking advantage of new technologies

Chromatography full scan HRMS Feasability Potential/limitations Data processing strategies Validation/application

Comprehensive analysis => big data...

Software/data processing development Data preprocessing/alignment Automated screening/detection modules HRMS libraries

Food safety stake holders: multiple perspectives

Yield & Quality

Sales & reputation

Price & Taste

Law & Enforcement

Research & Analysis data

https://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiF24nvivHOAhVJWhQKHWLvCO4QjRwIBw&url=https://www.dreamstime.com/stock-photo-store-manager-supermarket-smiling-holding-his-thumbs-up-image44368724&psig=AFQjCNGmxDQt-5V9V9dqRn-XJm33Q4UTHg&ust=1472919465946021

http://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj6mvW0i_HOAhXEvRQKHZRiCiYQjRwIBw&url=http://www.fda.gov/ForConsumers/ConsumerUpdates/ucm185769.htm&bvm=bv.131783435,d.ZGg&psig=AFQjCNHI-SeMn0pZA6XXZs_cvFxN7SveKw&ust=1472919620479220

http://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj2nbqPjPHOAhULWxQKHUo1B1QQjRwIBw&url=http://www.consumerreports.org/cro/magazine/2014/08/natural-on-food-labels-is-misleading/index.htm&bvm=bv.131783435,d.ZGg&psig=AFQjCNGyOVR8kJyiy_wUEzVr2Tkcb2Yncw&ust=1472919817683972

http://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjh09PwjPHOAhWCvBQKHSlxDrgQjRwIBw&url=http://www.alamy.com/stock-photo/pesticide-sprays.html&bvm=bv.131783435,d.ZGg&psig=AFQjCNF-9CLknMMh9f7PS2cTu3LsH7ERAA&ust=1472919968496594

http://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjpjo2fj_HOAhVLsBQKHWw9BUkQjRwIBw&url=http://www.foodnavigator-asia.com/Policy/Chinese-authorities-taking-major-steps-to-improve-food-safety-standards&bvm=bv.131783435,d.ZGg&psig=AFQjCNG9nrHyjUIixaKQaA2Lp7OzLDOtLg&ust=1472920643434309

Legislation (EU)

Not regulated yet but ‘of potential concern’

Concerns of the future?

Documents

Chemical analysis and screening approaches in food safety ... · Chemical analysis and screening approaches in food safety control [email protected]