chemical analysis and screening approaches in food safety ... · chemical analysis and screening...
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Chemical analysis and screening approaches in food safety control
hans.mol@wur.nl
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....
Food safety in the media....
Food safety in the media....
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
Food safety in the media....
Food safety in the media....
Food safety in the media....
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
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
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Luke EtAc Quechers Myco ACN MeOH ACE
% o
f 170 p
esti
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toxin
s/p
lan
t to
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<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
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
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Time (min)
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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
m/z= 196.0879-196.0899 F: FTMS + p ESI Full ms [70.00-1000.00] MS QEx_150618_056
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
m/z= 308.15086-308.15394 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.28E4
m/z= 311.07934-311.08246 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 1.29E4
m/z= 916.52182-916.53098 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 9.93E4
m/z= 290.17365-290.17655 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.08E4
m/z= 336.17882-336.18218 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
RT: 0.00 - 25.02 SM: 7B
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Time (min)
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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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Time (min)
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1007.65
11.35
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8.54 8.57
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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
m/z= 308.15086-308.15394 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.28E4
m/z= 311.07934-311.08246 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 1.29E4
m/z= 916.52182-916.53098 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 9.93E4
m/z= 290.17365-290.17655 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.08E4
m/z= 336.17882-336.18218 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
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
0 5 10 15 20 25
Time (min)
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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
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
m/z= 308.15086-308.15394 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.28E4
m/z= 311.07934-311.08246 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 1.29E4
m/z= 916.52182-916.53098 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 9.93E4
m/z= 290.17365-290.17655 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.08E4
m/z= 336.17882-336.18218 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
sulfadimethoxine
RT: 0.00 - 25.02 SM: 7B
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Time (min)
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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
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
m/z= 308.15086-308.15394 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.28E4
m/z= 311.07934-311.08246 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 1.29E4
m/z= 916.52182-916.53098 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 9.93E4
m/z= 290.17365-290.17655 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.08E4
m/z= 336.17882-336.18218 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
m/z 916.5264 C46H77NO17
tylosine
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
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
m/z= 308.15086-308.15394 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.28E4
m/z= 311.07934-311.08246 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 1.29E4
m/z= 916.52182-916.53098 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 9.93E4
m/z= 290.17365-290.17655 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.08E4
m/z= 336.17882-336.18218 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
m/z 290.1751 C17H23NO3
atropine
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
m/z= 308.15086-308.15394 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.28E4
m/z= 311.07934-311.08246 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 1.29E4
m/z= 916.52182-916.53098 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 9.93E4
m/z= 290.17365-290.17655 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.08E4
m/z= 336.17882-336.18218 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
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
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
m/z= 308.15086-308.15394 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.28E4
m/z= 311.07934-311.08246 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 1.29E4
m/z= 916.52182-916.53098 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 9.93E4
m/z= 290.17365-290.17655 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
NL: 6.08E4
m/z= 336.17882-336.18218 F: FTMS {1,1} + p ESI Full ms [55.00-1000.00] MS Exa_120806_29
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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Ab
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1009.13 9.16
9.11
9.17
9.18
NL: 1.81E7
m/z= 202.08439-202.08641 F: FTMS + p ESI Full ms [135.00-1000.00] MS QEx_140918_102
NL: 7.50E5
m/z= 124.08630-124.08754 F: FTMS + p ESI Full ms2 150.00@hcd55.00 [50.00-225.00] MS QEx_140918_102
NL: 1.60E7
m/z= 202.08529-202.08731 F: FTMS + p ESI Full ms [135.00-1000.00] MS QEx_140918_102
NL: 3.09E7
m/z= 145.06406-145.06552 F: FTMS + p ESI Full ms2 150.00@hcd55.00 [50.00-225.00] MS QEx_140918_102
XICs MEW ±5 ppm
Full
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RT: 8.15 - 10.15 SM: 5G
8.5 9.0 9.5 10.0
Time (min)
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Ab
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1009.13 9.16
9.11
9.17
9.18
NL: 1.81E7
m/z= 202.08439-202.08641 F: FTMS + p ESI Full ms [135.00-1000.00] MS QEx_140918_102
NL: 7.50E5
m/z= 124.08630-124.08754 F: FTMS + p ESI Full ms2 150.00@hcd55.00 [50.00-225.00] MS QEx_140918_102
NL: 1.60E7
m/z= 202.08529-202.08731 F: FTMS + p ESI Full ms [135.00-1000.00] MS QEx_140918_102
NL: 3.09E7
m/z= 145.06406-145.06552 F: FTMS + p ESI Full ms2 150.00@hcd55.00 [50.00-225.00] MS QEx_140918_102
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
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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Fortification level (µg/kg)
Method performance per analyte
0
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60
80
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
Example semi non-target detection
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
Example semi non-target detection
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 450.00@hcd55.00 [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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10011.75
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 450.00@hcd55.00 [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 450.00@hcd55.00 [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
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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
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’
Identification of the ‘unknown’
Identification of the ‘unknown’
Manual evaluation of the options.....
Structural analogue of sildenafil
Identification of the ‘unknown’
=> 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
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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
m/z= 196.0879-196.0899 F: FTMS + p ESI Full ms [70.00-1000.00] MS QEx_150618_056
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
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
- 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
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
0.46
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
m/z= 290.17365-290.17655 F: FTMS {1,1} + p ESI Full ms [60.00-1000.00] MS Exa_130322_090
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
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
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
Exa_130610_050 #14-40 RT: 0.20-0.56 AV: 27 NL: 4.71E6T: FTMS {1,1} + p ESI Full ms [60.00-1000.00]
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
Exa_130610_050 #14-40 RT: 0.20-0.56 AV: 27 NL: 6.76E5T: FTMS {1,1} + p ESI Full ms [60.00-1000.00]
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
Legislation (EU)
Not regulated yet but ‘of potential concern’
Concerns of the future?
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