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

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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

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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 glabinfo@govtlab.gov.hk

Thank You

The End