Analysis of Pesticide Residues in Food - · PDF file2 nd technical seminar OC–...
Transcript of Analysis of Pesticide Residues in Food - · PDF file2 nd technical seminar OC–...
Analysis of Pesticide Residues in Food
Dr C H Lam
Chemist
Government Laboratory
HKSAR
1st technical seminar
Analysis of 20 Organochlorine (OC) Pesticide Residues in Fruit and Vegetables
2nd technical seminar
OC – 35 Organochlorine pesticides (chlorinated pesticides)
OP – 44 Organophosphorus pesticides PY – 8 Pyrethroid pesticides Carb – 16 N-methyl carbamate pesticides 4 classes of pesticides
2nd technical seminar
Sample type No. of sample (2007)
Testing scope
Fruit and vegetables ~ 1000
Organochlorine orOrganophosphorus or
Pyrethroid orN-methyl Carbamate
Other food types ~ 500eg. Cereal and cereal products
eg. Meat
eg. Oil and fat
eg. Beverages
High starch foodHigh protein foodHigh fat foodLiquid
Testing scope
1. Organochlorine pesticidesTesting scope – 35 organochlorine pesticides
Alpha-BHC Aldrin
Beta-BHC Dichlorbenil
Gamma-BHC Dieldrin
Delta-BHC Dicofol
o,p’-DDE Alpha-Endosulfan
p,p’-DDE Beta-Endosulfan
o,p’-DDD Endosulfan Sulphate
p,p’-DDD Endrin
o,p’-DDT Heptachlor Epoxide
p,p’-DDT Heptachlor
POPs
1. Organochlorine pesticidesTesting scope –35 organochlorine pesticides
Hexachlorobenzene Chlorfenapyr
Methoxychlor Chlorothalonil
Mirex Quintozene
Procymidone Pentachloroaniline
Tetradifon Pentachlorophenyl sulphide
Trifluralin
Vinclozolin
cis-Chlordane
trans-Chlordane
Oxychlordane
Fungicide
2. Organophosphorus pesticidesTesting scope – 44 organophosphrous pesticides
Acephate Disulfonton
Azinphos-methyl Edifenphos
Carbophenthion Ethion
Chlorfenvinphos Ethoprophos
Chlorpyrifos Fenamiphos
Chlorpyrifos-methyl Fenchlorphos
Coumaphos Fenitrothion
Crufomate Fensulfothion
Diazinon Fenthion
Dichlorvos Isocarbophos
Dimethoate Isofenphos
敵敵畏
2. Organophosphorus pesticidesTesting scope – 44 organophosphrous pesticides
Malathion Phosmet
Methamidophos Phosphamidon
Methidathion Pirimiphos-Methyl
Mevinophos Profenophos
Monocrotophos Prothiophos
Omethoate Pyrazophos
Parathion-ethyl Quinalphos
Parathion-methyl Sulfotep
Phenthoate Tetrachlorvinphos
Phorate Triazophos
Phosalone Trichlorfon
甲胺磷
3. Pyrethroid pesticides Testing scope – 8 pyrethroid pesticides
Bifenthrin
Cyfluthrin
Cyhalothrin
Cypermethrin
Deltamethrin
Fenpropathrin
Fenvalerate
Permethrin
R2 R1
except fenvalerate
4. N-methyl carbamate pesticides Testing scope – 16 N-methyl carbamate pesticides
Aldicarb Ethiofencarb
Aldicarb sulfone Isoprocarb
Aldicarb sulfoxide Methiocarb
Bendiocarb Methomyl
Butocarboxim Metolcarb
Carbaryl Oxamyl
Carbofuran Promecarb
3-hydroxycarbofuran Propoxur
R
Me
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Sample preparation prior to analysis
Storage Fruit and vegetables 4oC Meat -20oC Cereal and cereal products room temperature (away from direct sunlight)
Our experience:Beware of using pesticides
Our experience:Enough room for sample storage (pre & post-analysis)
Classification of Food CommoditiesCodex Alimentarius
Vol. 2, Section 2: Pesticides Residues in Food, Codex Classification of Foods & Animal Feeds. [1]
Vol. 2A, Section 2.1: Analysis of Pesticides Residues: Portion of the Commodities to Which Codex MRLS Apply & Which is Analysed.[2]
Classification of Food Commodities
Type[1] Group[1] Analytical portion [2]
Vegetables Leafy vegetables “Whole commodity after removal of obviouslydecomposed or withered leaves.”
Fruits Stone fruits “Whole commodity after removing stems & stones but the residue calculated & expressed on the whole commodity without stem”
Grasses Cereal grains “Whole commodity”
Mammalian products
Meat “Whole commodity.”
Testing
Testing procedures
Organochlorine& Pyrethroidpesticides
Sample homogenization
Extraction
GPC cleanup
Florisil column cleanup
Instrumental analysis
Testing procedures
Organophosphorus& N-methyl carbamatepesticides
Sample homogenization
Extraction
GPC cleanup
Florisil column cleanup
Instrumental analysis
50-100g fruit and vegetables
Sample homogenization
Our experience:Sample size 1. Reporting limit2. Sample cleanup3. Sample homogeneity
25-50g high starch food (cereal & cereal products)10-25 g high protein food (eg. meat)1-2 g high fat food (eg. oil & fat) (direct dissolve in solvent GPC cleanup)50g liquid (eg. beverages)
Extraction
Our experience:Anhydrous Na2SO4Incomplete removal of water low recovery of polar pesticides
200mL ethyl acetate for fruit and vegetables300g anhydrous Na2SO4 Surrogate standard solution
Homogenize for ~5 min
200mL ethyl acetate for cereal and cereal productsNo anhydrous Na2SO4 Surrogate standard solution
200mL ethyl acetate:petroleum ether (7:3) for meat60g anhydrous Na2SO4 Surrogate standard solution
10mL cyclohexane:dichloromethane (1:1) for oil and fat (in vol. flask)No anhydrous Na2SO4 Surrogate standard solution
200mL ethyl acetate for beverages300g anhydrous Na2SO4 Surrogate standard solution
Surrogate standard solution
OC & PY – PCB 209 (decachlorobiphenyl) OP – TPP (tri-phenyl phosphate) Carb – 4-Bromo-3,5-dimethylphenyl N-methyl
carbamate (BDMC)
Extraction
Centrifuge for ~10 min
Filter the supernatant solution (through anhydrous Na2SO4)Record the volume of the extract collected
Extraction
Concentrate to just dryness using a rotary evaporator
Our experience:Loss of volatile pesticide
Reconstitute in cyclohexane:dichloromethane(1:1)
Anhydrous Na2SO4 drying Filter
For meat, fat content is needed to be determined.Transfer exact amount of extract to a pre-weighed beaker.Evaporate the solvent on a water bath.Dry to constant weight in 105oC oven
Gel permeation chromatography (GPC) cleanup
Size exclusion Removal of large molecule matrix Oils & pigments
Our experience:Incomplete removal of non-volatile materials deterioration of system performance
Gel permeation chromatography (GPC) cleanup
GPC column 60cm x 2.5cm id glass column packed with 60g 200-400 mesh
Bio-beads SX-3 resin in cyclohexane :dichloromethane (1:1)
Mobile phase cyclohexane : dichloromethane (1:1)
Flow rate 5mL/min.
Sample volume 5mL
Collection time 24-60min.
Gel permeation chromatography (GPC)
Concentrate to just dryness using a rotary evaporator
OC Florisil column cleanup GC-mECDPY Florisil column cleanup GC-mECDOP GC-FPDCarb HPLC-FLD
Florisil column cleanup
Solid phase extraction Removal of polar interference most OP & Carb pesticides can not be recovered
from Florisil column
Our experience:OP – GC-FPD Carb – HPLC-FLDBetter selectivity
Florisil column cleanup
Florisil column Florisil 500mg
1. condition 10mL 15% ethanol in petroleum
ether
2. sample load 3. elution
25mL diethyl ether in petroleum
ether for OC & PY
12.5 mL 50% dichloromethane/0.35% acetonitrile/49.65% hexane (v/v/v) and 12.5 mL 50% dichloromethane / 1.5% acetonitrile / 48.5% hexane (v/v/v)for chlorothalonil (PAM)
Our experience:Chlorothalonil Separate analysis
Florisil column cleanup
Concentrate to just dryness under N2 stream
Instrumental analysis OC GC-mECDPY GC-mECDOP GC-FPDCarb HPLC-FLD
GC – mECD GC conditions:-
Column : DB-1701, 30m, 0.25-mm i.d., 0.25-µm film thicknessHP-5, 30m, 0.25-mm i.d., 0.25-µm film thickness
Dual column analysis
Oven temp. : 100oC 10oC/min. 280oC (25min.)
Injection mode : splitless
Injection vol. : 1 µL
Injector temp.: 200 ºC.
Detector temp.: 300 ºC
Carrier gas flow: 1 mL/min. nitrogen
OC & PY
Our experience:Dual column -Eliminate false positive
Organochlorine pesticides (2 Groups)
Pyrethroid pesticides
Pyrethroids (multi-peaks) isomers R2 R1
GC – FPD (P filter) GC conditions:-
Column : DB-17, 30m, 0.53mm i.d., 1µm film thickness
Oven temp. : 140oC 7oC/min. 260oC (10min.)
Injection mode : splitless
Injection vol. : 1 µL
Injector temp.: 210 ºC.
Detector temp.: 230 ºC
Carrier gas flow: 10 mL/min. helium
OP
Organophosphorus pesticides (4 groups)
Our experience:Analyte protectant3-ethoxy-1,2-propanediol
Organophosphorus pesticides (4 groups)
HPLC – FLD HPLC conditions:-
Column : C18 column, 5mm, 250mm x 4.6mm (30oC)
Solvent A: purified water; Solvent B: acetonitrile
Flow rate: 0.8mL/min.
Gradient elution program:
Carb
Time/min. % A % B0 86 14
60 34 6665 86 14
Detector conditions:- Excitation wavelength: 339nm
Emission wavelength: 445nm
HPLC – FLD Post column reactor conditions:-
Hydrolysis:
Hydrolysis reagent flow rate: 0.5mL/min.
0.05N NaOH in purified water
Temperature: 80oC
Derivatization:
Derivatization reagent flow rate: 0.5mL/min.
100 mg o-phthalaldehyde(OPA) in 10 mL methanol and transfer to a 1 L volumetric flask and followed by adding 50 mL 2-mercaptoethanol(MERC). Dilute to the mark with 0.05 M sodium tetraborate solution. (PAM)
Temperature: room temperature
Carb
R
Me
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N-methyl carbamates are hydrolyzedto methylamine under alkaline condition
Methylamine is reacted with o-phthalaldehyde (OPA) & 2-mercaptoethanol(MERC) to form a fluorophore
N-methyl carbamate pesticides (2 groups)
Reporting limits
OC – 0.01 mg/kg OP ,PY & Carb – 0.1 mg/kg
Confirmation
Confirmation
GC-MS (EI & NCI) Applicable to all 4 classes of pesticides (except aldicarb,
aldicarb sulfon, aldicarb sulfoxide)
LC-MS/MS (ESI+) Applicable to N-methyl carbamate pesticides
Column : HP-5MS, 30m, 0.25-mm i.d., 0.25-µm film thickness
Oven temp. : 90oC(0.6min.) 20oC/min. 200oC (6min.) 20oC/min. 260oC (5min.)
Injection mode : splitless
Injection vol. : 2 µL
Injector temp.: 200 ºC
Transfer line temp.: 280 ºC
Carrier gas flow: 0.9 mL/min helium
MS mode: EI
Scan range : m/z of 3 monitoring ions for each pesticide
(SIM mode)
GC-MS
OC, OP & PY
Column : HP-5MS, 30m, 0.25-mm i.d., 0.25-µm film thickness
Oven temp. : 80oC(1min.) 10oC/min. 260oC (5min.)
Injection mode : splitless
Injection vol. : 2 µL
Injector temp.: 150 ºC
Transfer line temp.: 280 ºC
Carrier gas flow: 0.9 mL/min helium
MS mode: EI
Scan range : m/z of 3 monitoring ions for each pesticide
(SIM mode)
GC-MS
Carb
Our experience:Low injector temperature
GC/MS : Chromatogram (SIM)
Column : HP-5MS, 30m, 0.25-mm i.d., 0.25-µm film thickness
Oven temp. : 100oC 10oC/min. 280oC (10min.)
Injection mode : splitless
Injection vol. : 2 µL
Injector temp.: 250 ºC
Transfer line temp.: 280 ºC
Carrier gas flow: 1 mL/min helium
Reagent gas: methane
MS mode: NCI
Scan range : m/z of 1-3 monitoring ions for each pesticide
(SIM mode)
GC-MS
OC & PY
Our experience:Negative CI -More sensitive for some pesticideeg. endrin, heptachlor epoxide
GC/MS : Chromatogram (SIM)
LC-MS/MS HPLC-MS/MS conditions:-
Column : C18 column, 5mm, 150mm x 2.1mm (room temperature)
Solvent A: 2mM ammonium formate and 3.6mM formic acid in purified water
Solvent B: 2mM ammonium formate and 3.6mM formic acid in 95% acetonitrile
Flow rate: 0.2mL/min.
Gradient elution program:
MS mode: ESI+
Monitor 2 MRM pairs
Time/min. % A % B0 95 52 95 5
16 30 7016.2 95 520 95 530 95 5
LC-MS/MS
Confirmation
Relatively intensity(% of base peak)
EI GC-MS(relative)
CI GC-MS, LC-MS/MS (relative)
>50% ±10% ±20%
>20% to 50% ±15% ±25%
>10% to 20% ±20% ±30%
< 10% ±50% ±50%
Competency
Interlaboratory comparison - FAPAS (http://www.fapas.com)- CNAS / China National Accreditation Service for
Conformity Assessment (http://eng.cnas.org.cn) Certified reference material - National Measurement Institute / NIM (Australian
Government) Selected OP, OC and pyrethroid pesticides in pureed tomato
- Community Bureau of Reference / Organochlorine Pesticides in Animal Feed
Analytical methods under development
simultaneous qualitative and quantitative multi-class pesticides determination
International Reference / 國際參考
Analysis of Pesticide Residues: Recommended Methods, Codex Stan 229
Pesticide Analytical Manual (PAM), FDA
Official Methods of AOAC INTERNATIONAL, 970.52
水果和蔬菜中500種農藥及相關化學品殘留的測定-氣相色譜-質譜法, 中華人民共和國國家標準 GB/T 19648-2006
水果和蔬菜中405種農藥及相關化學品殘留的測定-液相色譜-串聯質譜法, 中華人民共和國國家標準 GB/T 20769-2006
Analytical Methods for Residual Compositional Substances of Agricultural Chemicals, Feed Additives and Veterinary Drugs in Food, Japan official method
Publications Application notes
Method validation
Analysis of Pesticides: Guidelines on Good Laboratory Practice in Residue Analysis, Codex CAC/GL40-1993, Rev.1 – 2003
Method Validation & Quality Control Procedures for Pesticide Residues Analysis in Food & Feed, SANCO/2007/3131, EU
Guidelines on the Use of MS for Identification, confirmation & Quantitative Determination of Residues, Codex CAC/GL 56-2005
Commission Decision, 2002/657/EC, implementing Council Directive 96/23/EC concerning the performance of analytical methods & the interpretation of results
Food Standards Agency Information Bulletin on Methods of Analysis & Sampling for Foodstuffs, Food Standards Agency, No. 42, Jan. 2004
EURACHEM/CITAC guide, Quantifying uncertainty in analytical measurement, 2nd ed.
My experience:
International Reference
Website:
www.codexalimentarius.net www.cfsan.fda.gov http://ec.europa.eu www.food.gov.uk www.epa.gov
Contact Us
Address Hong Kong Government Laboratory
7/F, Ho Man Tin Government Offices88, Chung Hau Street, Ho Man TinKowloon, Hong Kong
E-mail Address [email protected]
Thank You
The End