Efficient Monitoring Technique in
Food Safety Applications
Natchanun Leepipatpiboon, Urairat Koesukwiwat,
Thanatchaporn Semathong, Steven J. Lehotay
Efficient Monitoring Technique in Food
Safety Applications
Natchanun Leepipatpiboon, Urairat Koesukwiwat,
Thanatchaporn Semathong, Steven J. Lehotay
Food Safety
Food laws enforcement
Efficient analytical methods will be needed to monitor chemical
residues in foods and other sample types
2
Requirements and Expectations
Requirements
•
•
•
•
High degree of identification/confirmation
Low limit of detection
Accurate result
Robustness
Document No.
SANCO/12495/2011
Implemented by
01/01/2012
Regulations
Positive List System
for Agricultural
Chemical Residues in
Foods
Codex Maximun
Residue Levels
(MRLs)
(758 compounds)
>400
compounds
Other
features
required
•
•
•
•
Wide scope of analyte and matrix
Short analysis time
Affordable cost
Simple to perform
4
Routine Monitoring
Streamlining method and
Robust method for
“High sample throughput”
Wide range of analytes and
Various type of matrices ,
fruits, vegetables, meet etc. with
Fast and low operation cost
How we can meet all needs?
5
Analytical Method
Sample
preparation
LLE SPE GPC SPME SFC
etc.
Instrumental
Analysis
GC HPLC
Data analysis
& Process
Report
Chemical Residues in Foods
Qualification and Quantitation
3
What’s QuEChERS?
Quick – Easy – Cheap – Effective – Rugged – Safe
Streamlines sample preparation of pesticide residues in foods
1. Sample communition
2. Extraction (MeCN, EtOAc)
pesticides
+ interferants
4. dispersive-SPE
3. Partitioning
with salts
(MgSO4 + NaCl)
clean-up
(MgSO4, PSA, C18,
GCB, etc.)
7
Dispersive-SPE Clean-up
d-SPE = dispersion of the sorbent
in the sample extract to retain
matrix interferants, but not
analytes
• No needed: SPE cartridge/manifold
extra apparatus
evaporation/concentration
skill
• Fast and easy procedure
• Less sorbent used
• Low cost
retained matrix
interferants
interested
pesticides
• Lost half of final extract volume to the sorbent
• Filtration (if needed)
Solid Phase Extraction
8
History of QuEChERS
for pesticides residue in fruits and vegetables
AOAC 2007.01
Original
EN 15662
acetate-buffered
unbuffered
citrate-buffered
(2007)
(2003)
(2008)
10 g sub sample
10 g sub sample
10 g sub sample



10 mL 1% HOAc in
MeCN
10 mL MeCN
10 mL MeCN


0.4 g/mL anh.MgSO4
0.1 g/mL NaCl
0.4 g/mL anh.MgSO4
0.1 g/mL NaCl
0.1g/mL Na3Cit2H2O
0.05 g/mL Na2Cit1.5H2O

0.4 g/mL anh.MgSO4
0.1 g/mL NaOAc

150 mg/mL anh.MgSO4
50 mg/mL PSA

150 mg/mL anh.MgSO4
25 mg/mL PSA

150 mg/mL anh.MgSO4
25 mg/mL PSA
9
J. AOAC Int. 90 (2007) 485
J. AOAC Int. 86 (2003) 412
Anal. Bioanal. Chem. 389 (2007) 1697
Comparison of 3 QuEChERS Methods
Peas
Apple-blueberry mixture
Limes
31 different pesticides using LCMS/MS and GCToF MS
The preference used is depending on analytes and matrices.
8
QuEChERS for Difficult Compounds
Phenoxy acids:
!!
mostly used in Thailand
 low MRL 0.5 mg/kg
 polar  high water solubility
 toxic derivatizing agents for GC
 laborious, solvent
Rice: dry, high fatty acids
QuEChERS for High-Fat Commodities
Flaxseeds
Peanuts
GPC for cleanup:
 High lipids




 High fatty acids
 Dry matrices
 Starch
Doughs
Currently use in Cereals-Producing
Companies in USA
solvent consumption
time consuming
high operation cost
laborious
Analytical Method
Sample
preparation
LLE SPE GPC SPME SFC
etc.
Instrumental
Analysis
GC HPLC
Data analysis
& Process
Report
Chemical Residues in Foods
Qualification and Quantitation
3
172 Pesticides in 14 min by UHPLC-MS/MS
2 transitions/pesticide
344 transitions
Dwell time: 10 ms
Interchannel delay: 10 ms
Column: Acquity UPLCTM BEH C18, 2.1 x 150mm, 1.7μm
Flow rate: 0.45 ml/min
Temperature: 65°C
Slide adapted from André de Kok, VWA-Amsterdam, The Netherlands
Determination Step
The easiest ways to reduce GC analysis time
• less viscous carrier gas
• shorter column
• high flow rate
• wider column
• rapid temperature programming
But!
 separation efficiency
 sample capacity
 sensitivity
need new instrument design
co-eluted analytes
Mastovska and Lehotay, J. Chromatogr. A 1000 (2003) 153-180.
15
LP-GC/MS
Low-Pressure Gas Chromatography/Mass Spectrometry
Restriction capillary column
Inlet
Wider analytical column
Mega-bore
column
(0.53 mm i.d. vs. 0.25 mm i.d.)
(10 m  0.53 mm,
1 µm film)
Restriction
capillary
(3 m  0.15 mm,
non-coated)
Shorter analytical column
(10 m vs. 30 m)
c
MS
Restrictor Connector Analytical column
LP-GC column set up
Thicker film stationary phase
(1 m vs. 0.25 m)
Vacuum outlet (from MS)
High gas flow rate
Fast temperature program
(10-20C/min vs. up to 70-100C/min)
Traditional GC column: 30 m  0.25 mm i.d.  0.25 m df , Run time: 30 min
16
Experimental and results
Inlet
LP-GC/MS Features
Restriction capillary
No special GC/MS instrument
or inlet needed
 Speed
 Sample capacity
 Elution temperature
 Sensitivity (S/N ratio)
Restriction
capillary
 Degradation of thermal
labile pesticides
 Peak tailing
Megabore column
 Separation efficiency
(but compensation by MS)
17
Experimental and results
Traditional GC/TOF MS
1e+007
8e+006
6e+006
25 min
4e+006
2e+006
Time (s)
600
800
1000
1200
1400
1600
1800
Much faster (4-fold) and more sensitive
LP-GC/TOF MS
2.25e+007
2e+007
1.75e+007
1.5e+007
1.25e+007
1e+007
6 min
7.5e+006
5e+006
2.5e+006
Time (s)
100
150
200
250
300
350
400
450
500
Full-scan ion chromatograms of 34 pesticide standards
18
Experimental and results
Traditional
GC/TOFMS
FWHM = full-width at half-maximum
LP-GC/TOFMS
Wh = 2.768 s
Wh = 1.519 s
Wh = 2.739 s
Wh = 1.101 s
Wh = 6.847 s
Wh = 3.713 s
19
Traditional GC-MS/MS method
27.5 min
New GC-MS/MS method
Much faster (4-fold) and more sensitive
 7 min
Full-scan ion chromatograms of >150 pesticide standards
High Throughput Analysis
+
+

Single and effective method and

Multi-residue method for

Wide range of pesticides and

Various fruits and vegetables with

Fast and low operation cost
QuEChERS and LP-GC/MS
15
Method Validation
updated QuEChERS (unbuffered vs. acetate-buffered with d-SPE vs. DPX) for
150 pesticides (+ 3 I.S./QC compounds) at
3 spiking levels (25, 100, 400 ng/g) with
5 replicates at each level + calibration standards in
water
high sugar
carbohydrate + fat
acid
high chlorophyll
1 hr for sample preparation
50 injections per day and 8 hrs sequence per day
4e+006
3.5e+006
3e+006
2.5e+006
2e+006
1.5e+006
1e+006
500000
Time (s)
150
200
250
300
350
400
20
Method Validation
Recovery:
70-110% for trace level
high fatty acids
in potato
RSD:
20% for trace level
ToF MS limitations
 sensitivity
 selectivity
 high detection limit
21
Experimental and results
LP-GC/MS-MS Conditions
Injection
Triple-quadrupole
MS/MS
Restrictor
Analytical column
3 m  0.15 mm i.d.
non-coated
GC: Agilent 7890A
Rti-5ms
10 m  0.53 mm i.d.  1 µm df
Flow rate: Helium at 20 psi constant
Injection: 5 µL PTV (Multimode Inlet, MMI) with sintered-glass liner (Agilent P/N 5190-1426)
Inlet: 80⁰C initial for 0.31 min to 320⁰C at 420⁰C/min
Oven: 70⁰C (1.5 min), 80⁰C/min to 180⁰C, 40⁰C/min to 250⁰C, 70⁰C/min to 290⁰C (4.3 min)
MS/MS: Agilent 7000A and 7000B triple-quadrupole
EI -70 eV, 250⁰C transfer line, 320⁰C ion source,
3 min solvent delay
Total run time: 9.5 min for 170 pesticides
24
LP-GC/MS-MS
Updated acetate-buffered QuEChERS
Cantaloupe
Broccoli
Sweet potato
Lemon
(water)
(chlorophyll)
(fatty acids)
(acid)
100% of
detected
analytes
>170 pesticides + 2 I.S. + QC
LP-GC/MS-MS
GC: Agilent 7890
MS-MS: Agilent 7000A triple-quadrupole
Method optimization:
 injection
 inlet temperature
 oven temperature
 specific MS/MS conditions
 matrix effects
High selectivity and sensitivity
LOD 10 ng/g within 9.7 min
22
26
Organochlorine
(46 cpns)
Alachlor
Aldrin
Alpha-BHC
Azaconazole
Benfluralin
Benoxacor
Beta-BHC
Bromophos-ethyl
Bromophosmethyl
Bromopropylate
Bupirimate
Butachlor
Cafentrazoneethyl
Chlordane-cis
Chlordane-trans
Chlorfenapyr
Chlorfenson
Chloroneb
Chlorthaldimethyl
Clomeprop
Delta-BHC
Dicloran
Diledrin
Endosulfan
sulphate
Endosulfan-alpha
Endosulfan-beta
Endrin
Ethalfluralin
Fipronil
Gamma-BHC
HCB
Heptachlor
Heptachlorepoxide
Mefenpyr-diethyl
Methoxychlor
Mirex
p,p’-DDD
p,p’-DDE
p,p’-DDT
Pyrifenox
Tecnazene
Tetradifon
Thiazopyr
Triallate
Trifluralin
Vinclozolin
Organophosphate
(45 cpns)
(E)-Dimethylvinphos
(Z)-Chlorfenvinphos
Pyrethoids
Butamifos
(9 cpns)
Cadusaphos
Carbophenothion
Chlorpyrifos
Bifenthrin
Chlorpyrifos-methyl
Cyfluthrin (I-IV)
Cyanophos
Cyhalothrin (I+II)
Demeton-S-methyl
Cypermethrin (I-IV)
Diazinon
Deltamethrin (I+II)
Dichlorfenthion
Fenpropathrin
Dichlorvos
Fenvalelate (I+II)
Disulfoton
Permethrin-cis
EPN
Permethrin-trans
Ethion
Ethoprophos
Phorate
Fenamiphos
Piperophos
Fenchlorfos
Pirimiphos-ethyl
Fensulfothion
Pirimiphos-methyl
Fenthion
Profenofos
Fonofos
Propetamphos
Formothion
Prothiophos
Iprobenfos
Pyridafenthion
Isazofos
Quinalphos
Isofenphos
Sulprofos
Malathion
Terbufos
Methacrifos
Thiometon
Parathion-ethyl
Tolclofos-Methyl
Parathion-methyl Triazophos
Phenthoate
Tribufos
Organonitrogen
(73 cpns)
(E)-Metominostrobin
Ametryn
Atrazine
Azoxystrobin
Benalaxyl
Benfuresate
Bromobutide
Bromobutide Metabolite
Buprofezin
Chlorpropam
Cimmethylin
Clomazone
Cyhalofop-butyl
Dichlobenil
Diclofop-methyl
Difenoconazole-cis,trans
Dimepiperate
Dimethametryn
Diniconazole
Dithiopyr
Esprocarb
Etofenprox
Fenclorim
Fenoxaprop-ethyl
Fenpropimorh
Flusilazole
Flutolanil
Furametpyr
Hexaconazole
Hydroxy Furametpyr
Imazalil
Iprodione
Isoprothiolane
Kresoxim-methyl
Mepronil
Metalaxyl
Metolachlor
Molinate
Oryzastrobin
Oryzastrobin metabolite
Oxadiazon
Oxyfluorfen
Paclobutazole
Penconazole
Pendimethalin
Picolinafen
Piperonyl butoxide
Pirimicarb
Pretilachlor
Prochloraz
Procymedone
Prometryn
Propachlor
Propham
Propiconazole-trans
Pyraflufen-ethyl
Pyributicarb
Pyriproxyfen
Pyroquilon
Resmethrin
Silafluofen
Simazine
Simeconazole
Simetryn
Tebuconazole
Terbutryn
Thiobencarb
Triadimefon
Triadimenol
Trifloxystrobin
Triticonazole
Uniconazole
Etobenzanid
27
Std at 0.1 mg/kg
GC/MSD
52.5 min
Azoxystrobin
Total ion chromatogram of pesticide standards
28
Traditional GC-MS/MS
29
LP-GC-MS/MS
Traditional GC-MS/MS
30
Experimental and results
2.5E+06
Traditional GC-MS/MS
2.0E+06
1.5E+06
1.0E+06
27.303 min
5.0E+05
0.0E+00
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
32
33
Much faster (4-fold) and more sensitive
3.5E+06
3.0E+06
LP-GC-MS/MS
2.5E+06
2.0E+06
1.5E+06
6.757 min
1.0E+06
5.0E+05
0.0E+00
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Counts vs. Acquisition Time (min)
Full-scan ion chromatograms of 220 pesticide standards
31
Experimental and results
Traditional
GC-MS/MS
3.0E+05
LP-GC-MS/MS
3.0E+05
2.0E+05
2.0E+05
FWHM = 0.020 min
Procymidone (283.0 >96.0)
FWHM = 0.036 min
1.0E+05
0.0E+00
13.7
1.0E+05
0.0E+00
13.8
13.9
4.3
14
4.4
4.5
4.6
3.0E+05
3.0E+05
2.0E+05
FWHM = 0.012 min
2.0E+05
Dichlorvos (109.0 > 79.0)
FWHM = 0.021 min
1.0E+05
1.0E+05
0.0E+00
0.0E+00
5.7
5.8
5.9
6
Chlorpyrifos (196.9 > 168.9)
FWHM = 0.037 min
2.0E+05
1.0E+05
1.0E+05
0.0E+00
12.3
12.4
12.5
12.6
Counts vs. Acquisition Time (min)
0.0E+00
FWHM = full-width at half-maximum
2.5
2.6
2.7
3.0E+05
3.0E+05
2.0E+05
2.4
FWHM = 0.019 min
4
4.1
4.2
4.3
Counts vs. Acquisition Time (min)
32
3.0E+05
Experimental and results
Traditional
GC-MS/MS
LP-GC-MS/MS
3.0E+05
2.0E+05
2.0E+05
FWHM = 0.019 min
Phosalone (182.0 > 111.0)
FWHM = 0.047 min
1.0E+05
0.0E+00
20.4
20.5
20.6
20.7
Counts vs. Acquisition Time (min)
1.0E+05
0.0E+00
5.5
2.0E+05
2.0E+05
1.0E+05
1.0E+05
Tebuconazole (249.9 > 125.0)
5.6
5.7
5.8
Counts vs. Acquisition Time (min)
FWHM = 0.044 min
0.0E+00
17.95
FWHM = 0.022 min
0.0E+00
18.05
18.15
18.25
1.0E+04
5.1
5.2
5.3
5.4
1.0E+04
FWHM = 0.029 min
Azoxystrobin (388.0 > 345.0)
FWHM = 0.05 min
0.0E+00
27.2
0.0E+00
27.3
27.4
27.5
6.6
6.7
6.8
6.9
MRM chromatogram in updated-QuEChERS extracts
Dichlorvos
Methamidophos
Omethoate
m/z 109  79
m/z 185  93
m/z 141  95
m/z 141  80
m/z 156  110
m/z 156  79
Dimethoate
Phosalone
Coumaphos
Azoxystrobin
m/z 125  79
m/z 143  111
m/z 182  111
m/z 182  75
m/z 362  109
m/z 362  226
m/z 344  329
m/z 344  172
0.01 mg/Kg
GC-MS/MS
LPGC-MS/MS
35
Sample preparation
Sample extraction:
based on the AOAC QuEChERS method
- 15 g of homogenized sample
- 15 mL of 1% acetic acid in MeCN
- 6 g anh. MgSO4 and 1.5 g NaOAc
Dispersive SPE clean-up:
150 mg anh. MgSO4 + 50 mg PSA + 50 mg C18 + 7.5 mg GCB
per 1 mL of extract
Experimental and results
Approximate Comparison
Traditional
approach
QuEChERS
and GC-MS
QuEChERS
and LP-GC/MS






Operation time



Hazardous waste



Operator skill



Method
Disposable material costs
(solvent, vial, tube, sorbent, etc.)
Extra apparatus for sample
preparation
(soxhlet, SPE, GPC, SFE,
evaporator, etc.)
Operation costs
(instruments, maintenance,
service contacts, etc.)

37
Conclusions
QuEChERS: fast and easy sample preparation method
UHPLC-MS/MS, GC-MS, LP-GC/MS(-MS): increase degree of
identification and confirmation
Wide scope of analysis!
LC- and GC-MS(/MS)
wide range of pesticides
many types of food (fruits, vegetables, meats, milk, etc.)
Low limit of detection
High sample throughput
Low operation cost per sample (40-50% reduction)
Less hazardous waste
Reliable and suitable for routine analysis
23
Thank you for your attention
39