Developing a Pesticide Analysis Program to Meet International

Food Trade Requirements:The Codex Alimentarius

Influence

Philip L. Wylie, Ph.D.

Agilent Technologies

Wilmington, DE USA

phil_wylie@Agilent.com

Presentation Outline

• Pesticide use around the world

• Pesticides and International Food Trade

• Codex Alimentarius

• Starting a Pesticide Monitoring Program for international food trade

• Where to get help & find requirements

• Your instrumentation investment - what equipment is needed

Pesticides Usage• More than 700 pesticides are registered for use WW

• About 2.2 billion kg of pesticide used each year WW

• 1995 WW pesticide sales = $29 billion

• Some very toxic pesticides are banned in many countries but may still be used in others:

– Endrin, DDT, lindane, aldrin, chlordane, and many others

• No standardization of Maximum Residue Limits (MRLs) in food

• Banned or highly restricted pesticides have been “dumped” in developing countries

Pesticide MRL Influence on International Food Trade

• Many countries have rigorous pesticide registration processes

• Many countries have created their own MRLs for pesticides based on local laws

• Within one country MRLs are often different on different food types (pear MRL may not be the same as onion MRL)

• MRLs are often different in different countries

•Differences can lead to Intentional or unintentional trade barriers

Comparison of Codex MRLs for Apples (ppm) to Some National MRLs

PesticideCaptan 25 10 3 3 5 25 Codex 25Carbaryl 5 5 3 3 1 10 1 10Dimethoate 1 5 1 1 2 2Fosetyl-al 1 75 USA 10Malathion 2 2 0.5 0.5 0.5 8 Codex 8Phosmet 10 1 1 2 10 2 10

* In some cases Taiwan uses Codex or US MRLs

Reference: Northwest Horticultural Council Export Manual

Available at: http://www.nwhort.org/exporttoc.html

Pesticide Related Trade Barriers

Pesticides used legally on a crop in an exporting country are not registered for use in the importing country - e.g. banana growers may use a safe pesticide but an importing country doesn’t need or use that pesticide

Pesticide Related Trade Barriers

Different MRLs - Residues at legal levels in exporting country are too high for the importing country

Pesticide Related Trade Barriers

Contamination from “extraneous” pesticides such as DDT that may be in the soil from previous use

Past DDT Spraying

Economic Trade Barriers

• Designed to protect farm products of the importing country

• May be disguised as health or safety measures

• May be a response to consumer misconceptions about the dangers

•Pesticide or drug MRLs could be set so low that it prevents imports

Fruit spoils when rejected by importer

United Nations Creates the Codex Alimentarius Commission

“Codex Alimentarius” is Latin for “Food Code”

• Created in 1961-3 by the United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO)

• Now has 165 Member Countries representing 97% of the world’s population

Codex Alimentarius Commission Goals

• To develop a compilation of scientifically-based standards, codes of practice and guidelines for the production, processing and shipping of food...

• In order “to protect the health of consumers and to ensure fair practices in the food trade”

• To “harmonize” food safety standards world wide

• To assist developing countries in meeting these standards

Organizations Associated with the Codex Alimentarius

• Joint FAO/WHO Meeting on Pesticide Residues (JMPR)

– JMPR is independent of the CODEX Commission

– JMPR recommends maximum residue limits (MRL)

• Joint FAO/WHO Expert Committee on Food Additives (JECFA)

– JECFA is independent of the CODEX Commission

– JECFA recommends limits for veterinary drug residues, food additives and other contaminants

• International Atomic Energy Agency (IAEA)

– FAO/IAEA Training and Reference Centre for Food and Pesticide Control (http://www.iaea.org/programmes/rifa/trc/index2.htm)

Codex Alimentarius:

Has developed - Food standards for 237 different commodities - 41 codes of hygienic or technological practice - 25 guidelines for contaminants - 3274 MRLs for pesticide residues

Has evaluated - 1005 food additives - 54 veterinary drugs - 185 different pesticides

Contents of the Codex Alimentarius

• Volume 1A - General requirements

• Volume 1B - General requirements (food hygiene)

• Volume 2A - Pesticide residues in foods (general texts)

• Volume 2B - Pesticide residues in foods (maximum residue limits)

• Volume 3 - Residues of veterinary drugs in foods

• Volume 4 - Foods for special dietary uses (including foods for infants and children)

• Volume 5A - Processed and quick-frozen fruits and vegetables

Contents of the Codex Alimentarius• Volume 5B - Fresh fruits and vegetables

• Volume 6 - Fruit juices

• Volume 7 - Cereals, pulses (legumes) and derived products and vegetable proteins

• Volume 8 - Fats and oils and related products

• Volume 9 - Fish and fishery products

• Volume 10 - Meat and meat products; soups and broths

• Volume 11 - Sugars, cocoa products and chocolate and miscellaneous products

• Volume 12 - Milk and milk products

• Volume 13 - Methods of analysis and sampling

Contents of Vol. 2A: “Pesticide Residues in Food - Methods of Analysis and Sampling”

• Recommended Methods of Sampling for the Determination of Pesticide Residues for Compliance with MRLs

• Recommended methods of analysis for pesticide residues

– Portion of commodities to which codex MRLs apply and which is analyzed

– Guidelines on Good Laboratory Practice in pesticide residue analysis

– Suggested analytical methods

Where to get the Codex Alimentarius

• Sales and Marketing Group Food and Agriculture Organization of the United Nations Viale delle Terme di Caracalla 00100 Rome, Italy Email : publications-sales@fao.org

• Some parts of the Codex Alimentarius can be viewed on the web at:

– http://www.codexalimentarius.net

Available in English, French, & Spanish

Imagine the Following Scenario in Your Country: Sunshine Island

• Sunshine Island grows a variety of tropical fruits and vegetables

• Exports are growing, which benefits the local economy

• But, sometimes shipments are rejected by the importer because of pesticide residues - it has become a costly problem

• Sunshine Island has not monitored pesticide residues in the past

• You must set up a laboratory to analyze your food to meet Codex Pesticide MRLs

You have Some Important Questions • What are the MRLs for pesticides in our export products?

• What extraction procedures should we use?

• What analytical techniques do we need?

• What instrumentation do we need?

• How do we validate our methods?

• Where can we get help in developing Standard Operating Procedures (SOPs)?

• Where can we get training?

Searching the Codex Database to Find all Pesticide MRLs for Papayas

http://apps.fao.org/CodexSystem/pestdes/pest_q-e.htm

Also available in Volume 2B of the Codex Alimentarius

Chinomethionat, Dithiocarbamates, and Prochloraz have Codex MRLs for Papayas

Pesticide

MRL Value (ppm)

Best Approach for Choosing Extraction and Analysis Methods

• Choose a method already in use by experienced pesticide analysts

– It will already be validated in at least one lab

• Make minor adaptations as needed for:

– differences in commodities

– differences in analytical equipment

• Validate the method in your laboratory

Where can we find Good Validated Methods?

• Florida Department of Agriculture and Consumer services

– J. Cook, M.P. Beckett, B. Reliford, W. Hammock, M. Engel (1999) J.

AOAC Int. 82, 1419-1435

• California Department of Food and Agriculture (www.cdfa.ca.gov)

– Multiresidue Screen for Pesticides in Fruits and Vegetables (1995)

California Department of Food and Agriculture, Sacramento, CA, USA

summary 1-2

– S.M. Lee, M.L. Papathakis, H.M.C. Feng, G.C. Hunter, J.E. Carr (1991)

Fresenius J. Anal. Chem. 339, 376-383

Where can we find Good Validated Methods?

• Ministry of Public Health, Welfare and Sport, The Netherlands

– Analytical Methods for Pesticide Residues in Foodstuffs, 6th ed.

(1996) General Inspectorate for Health Protection Ministry of Public

Health, Welfare and Sport (The Netherlands)

• Pesticide Analytical Manual (PAM)

– U. S. Food and Drug Administration

Center for Food Safety and Applied Nutrition

Office of Plant and Dairy Foods and Beverages

1994; Updated October, 1999

– Can download from the WWW at:

> http://vm.cfsan.fda.gov/~frf/pami1.html

– Includes a lot of basic information on chromatography

A Typical Fruit & Vegetable Extraction Procedure

This procedure is used by the Florida Department of Agricultureand Consumer Services as described in the following journal article:

J. Cook, M.P. Beckett, B. Reliford, W. Hammock, M. Engel (1999) J. AOAC Int. 82, 1419-1435

Florida Department of Food and Agriculture Extraction Steps

Weigh 50 g Homogenized Fruit/Vegetable Sample into 250-mL Polyethylene (PE) Bottle

Acetonitrile Extract

Add spiking solution and 100 mL CH3CN, cap & shake 3 min

Filtered Acetonitrile Extract

Gravity filtration

C-18 SPE under N2 pressure; Collect in 250 mL PE bottle containing 10 g NaCl

Extract & NaCl in Bottle

Shake 2 min; Centrifuge 2 min @ 1000 rpm; Decant into 70-mL SPE reservoir; Drain off H2O layer

Acetonitrile Extract Separated from H2O

Florida Department of Food and Agriculture Extraction Steps

Acetonitrile Extract Separated from H2O

Collect 15 mL in 100-mL BeakerCollect 15 mL in 100-mL Beaker

P Fraction C Fraction

Aminopropyl SPE-Cleaned Extract

Aminopropyl SPE on 4 mL of CH3CN extract; Elute with 3% methanol in acetone

Evaporate using N2 just to dryness; Add 1 mL MeOH; Filter into 2-mL ALS vial

Final Extract for Carbamate Analysis

HPLC/Fluorescence or HPLC/MS

Florida Department of Food and Agriculture Extraction Steps

GC Analysis (N, P, S Selective Detectors or GC/MS)

15 mL CH3CN Extract (P Fraction)

Evaporate to ~2 mL on steam bath; Air dry in hood

Dry Extract

Add 2 mL Acetone; Swirl to dissolve

Acetone Solution

Pour into 16 X 125 mm screw cap test tube; refrigerate >15 min to precipitate NaCl

Final Extract (P Fraction)

Florida Department of Food and Agriculture Extraction Steps

GC Analysis (Halogen-Selective Detector or GC/MS)

15 mL CH3CN Extract (C Fraction)

Evaporate to ~2 mL on steam bath; Air dry in hood

Dry Extract

Add 2 mL 85:15 hexane:acetone; Swirl to dissolve

Hexane:Acetone Solution

Florisil SPE; Rinse with 85:15 hexane:acetone; Collect in 15-mL graduated test tube

Florisil-Cleaned C Fraction in Hexane/Acetone

Reduce volume under N2; Adjust to 4 mL with isooctane

Almost All Pesticides Contain Heteroatoms (Oxygen is too common)

PNH

O

S OO

CH3

CH3

CH3

AcephateN

N N

NH NH

Cl

CH3

CH3 CH3

Atrazine

N+

N+

N

O-

O-

O

O

F

F

F

CH3

CH3

Trifluralin

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Heptachlor

Element-selective GC detectors are commonly used for pesticide analysis

GC Detectors for Pesticide AnalysisDetector Responds to: Advantages Disadvantages

Nitrogen-Phosphorus Detector (NPD)

N and P Cost, SelectivityDoes not differentiate N from P;

Electron Capture Detector (ECD)

Best for halogens & nitro groups

Cost, Selectivity; Sensitivity (See disadvantages)

Lower response for mono & dihalogenated compounds; Partial selectivity

Flame Photometric Detector (FPD)

S or PCost; Selectivity; Sensitivity

Electrolytic Conductivity Detector (ELCD)

HalogensResponds to mono & dihalogenated compounds

Hard to use and maintain; Only responds to halogens; Sales & Service not available everywhere;

Halogen-Specific Detector (XSD)

Halogens

Selectivity; Responds to mono and dihalogenated compounds

Sales & Service not available everywhere

GC Detectors for Pesticide Analysis

Detector Responds to: Advantages Disadvantages

Mass Selective Detector (MSD)

Everything: It's both universal and selective

Selective for all compounds; Linear over required range; Easy to use; One gas required; Positive identification; Sensitivity

Initial cost (but it may actually be cheaper)

Analytical Techniques and Instrumentation for Pesticide Analysis

• Basic target pesticide analysis (entry solution)

– Dual column GC(s) with selective detectors

> ECD for organochlorine pesticides

> NPD for organonitrogen pesticides and organophosphorus pesticides

> FPD for organophosphorus pesticides

– 30-m X 0.25-mm X 0.25 um HP-5MS column (P/N 19091S-433) & a more polar column for confirmation

– LC with post-column derivitization and fluorescence detection (Pickering System)

> Analysis of carbamate pesticides

Analytical Techniques and Instrumentation for Pesticide Analysis

• Typical Capability of Laboratories in the US and Europe

– GC(s) with selective detectors

> ECD for organochlorine pesticides

> NPD for organonitrogen pesticides and organophosphorus pesticides

> FPD for organophosphorus pesticides

– GC/MSD for pesticide confirmation

– LC/MSD for carbamates & other non-volatile or sensitive pesticides

Advanced Pesticide Analysis

• Use Retention Time Locking (RTL) for GC & GC/MS

– 30-m X 0.25-mm X 0.25 um HP-5MS column (P/N 19091S-433) locked to Agilent pesticide method

• Benefits of RTL

– Use Agilent GC and GC/MS RTL Libraries for pesticide identification

– Lock all GC and GC/MS methods

> All GC & GC/MS analyses have same RT

> All runs match the RTL Library RTs

– Easier pesticide identification

– Easier pesticide confirmations by GC/MS

Is a GC/MSD Really More Expensive than GCs with Selective Detectors?

Dual Column GC/NPD

Dual Column GC/FPD

Dual Column GC/ECD

GC/MSD

orIt will depend on your samples, the pesticides you analyze, and what level of confirmation is required.

Canada Analyzes Pesticides using only GC/MS and LC/Fluorescence

Julie Fillion, François Sauvé, and Jennifer Selwyn, Multiresidue Method for the Determination ofResidues of 251 Pesticides in Fruits and Vegetablesby Gas Chromatography/Mass Spectrometry andLiquid Chromatography with Fluorescence Detection,

J. AOAC Int., 83 (3), 698-713and

J. AOAC Int., 78 (5), 1252-1266

Note: Some countries do not “recognize” this method because many pesticides are identified by only two ions

Help in Validating Your Methods

• Start with a method that has already been validated

• Use the following resource prepared by the Joint FAO/IAEA Expert Consultation on Method Validation

– Guidelines for single-laboratory Validation of Analytical Methods for Trace-level Concentrations of Organic Chemicals

– Available on the www at:> http://www.iaea.org/trc/

Help in Developing Standard Operating Procedures (SOPs)?

• The Pesticide Data Program of the US Department of Agriculture has published all of their SOPs on the www at:

– http://www.ams.usda.gov/science/pdp/index.htm

– SOPs for

> Administration of sampling & lab processes

> sampling procedures

> laboratory analysis

~ standards, laboratory operations, quality control, and data handling

– Includes 60 SOPs

Training for Pesticide Analysis

• Courses are offered by the UN Food and Agriculture Organization with The International Atomic Energy Agency

– Courses are offered on: pesticide residue analysis, mycotoxin analysis, & quality control of pesticide products

– Requires nomination by your government

– See: http://www.iaea.org/trc/

• The Natural Resources Institute offers– 13-week Pesticide Residue Analysis Training & related Courses at the University of

Greenwich

– See: http://www.nri.org/

• Agilent Technologies

– Ask your Agilent representative

Conclusions

• International food trade requires pesticide residue analysis

• Codex Alimentarius provides harmonized regulations for foods, including pesticide MRLs

• Agilent Technologies provides

– all of the required analytical instruments

> GC, GC/MS, LC, LC/MS, LC/MS/MS, ICP/MS, Columns, Supplies

– analytical methods based on Retention Time Locking

– RTL pesticide databases for GC and GC/MS

– expert consultation and training