updates on testing of pesticide residues in food on testing of... · pesticide residues in food...

Updates on Testing of Pesticide Residues in Food

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28 October 2016

Food Testing – Pesticide Residues Previous briefs available on websites of

Government Laboratory

Centre for Food Safety

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http://www.govtlab.gov.hk/ http://www.cfs.gov.hk/

Pesticide Residues in Food

Pesticides and Definition-related Compounds

International Trends on Analytical Performance Criteria

Optimization of Pesticide Residues Methods

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- Discussion Topics:

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Pyrethrins

Meptyldinocap / 2,4-DNOP / Dinocap

Carbendazim / Benomyl / Thiophanate-methyl


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Pyrethrins (MRL item 288)

Residue definition:

Total pyrethrins, calculated as the sum of pyrethrins 1 and 2,

cinerins 1 and 2, jasmolins 1 and 2, determined after calibration

with World Standard pyrethrum extract

What is “Pyrethrum” ?

What is “Pyrethrum extract”

What is “Total pyrethirns’ ?

What is “World Standard pyrethrum extract” (W.S.P.E) ?

Reference Standard for total pyrethrins analysis ?



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What is “Pyrethrum” ?

Pyrethrum flowers

(Chrysanthemum cinerariaefolium)

Reference : Greenhill, M., 2007, Pyrethrum production: Tasmanian success story, Chronica Horticulturae. 47(3)

http://www.botanicalra.com.au/

• Traditionally produced by

Kenya

• Now also from Tasmania,

Australia (Industrialized)

[McLaughlin Gormley King Company:

http://www.pyrethrum.com/ ]


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What is “Pyrethrum extract”?

“the material shall consist of the clear extracts or concentrates

of substances occurring naturally in pyrethrum flowers

(Chrysanthemum cinerariaefolium) and shall be free from visible

extraneous matter.” [Reference : Australian Pesticides and Veterinary Medicines Authority]

How to produce pyrethrum extract?

Harvest the pyrethrum (With ~15% moisture remained)

Pelletize the harvested material and extract by hexanes

Supercritical refining

Standardized to ~50% pyrethrins

[Reference : Greenhill, M., 2007, Pyrethrum production: Tasmanian success story,

Chronica Horticulturae. 47(3)]


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What is “World Standard pyrethrum extract” (W.S.P.E.) ?

Provided by Pyrethrum Board of Kenya (PBK)

[http://www.kenya-pyrethrum.com/]

PBK maintains W.S.P.E. specific to the year of harvest.

[Some results published on

Pyrethrum Post (ISBN:0048-6043)] [ Available from “Friends of Pyrethrum” http://www.friendsofpyrethrum.co.uk/]

“World Standard pyrethrum extract” (W.S.P.E.) is no longer

available. [Personal communications between presenter and Botanical Resources Australia,

2013]


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What is “Total Pyrethrins’ ?

Analyte CAS RN

Cinerin I 25402-06-6

Jasmolin I 4466-14-2

Pyrethrin I 121-21-1

Cinerin II 121-20-0

Jasmolin II 1172-63-0

Pyrethrin II 121-29-9

Pyrethrins 8003-34-7

Reference :

- AOAC Official Method 997.07

- ISO 765-2016

- Journal of Chromatographic Science, Vol. 39, November 2001


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How the Pyrethrins content assigned?

1) PBK Mercury Reduction Method (W.S.P.E.)

2) “Pyrethrin in Pesticide Formulations – Mercury Reduction

Method (I)” [AOAC Official method 936.05]

3) “Identification and quantitation of the six active compounds

in a pyrethrin standard” [J. AOAC Int 80 966-973 (1997)],

as cited in

3a) “N-octyl Bicycloheptene Dicarboximide (MKG 264),

Pyrethrins and Piperonyl Butoxide (PB) in Technical

Materials, Concentrates, and Finished Products”

[AOAC Official method 997.07]

3b) CIPAC handbook H (1998)

3c) WHO specification of pyrethrum (2009)

4) HPLC-DAD method published by Aventis Environmental

Sciences [Journal of Chromatographic Science, Vol. 39, November 2001]


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What are determined from these methods?

AOAC 936.05

(Alkaline hydrolysis

+ Mercury Reduction

by Deniges reagent

+ titration)

J AOAC 80(5) 966 (1997)

(Modified AOAC 936.05

hydrolysis, followed by

GC-FID)

HPLC-DAD method,

published by Aventis

Environmental Sciences

(2001)

Cinerin I Cinerin I

Pyrethrins I (~ 60%) Jasmolin I Jasmolin I

(ie chrysanthemates) Pyrethrin I Pyrethrin I

Cinerin II Cinerin II

Pyrethrins II (~ 40%) Jasmolin II Jasmolin II

(ie pyrethrates) Pyrethrin II Pyrethrin II

Material usually contains

20-50 % Total Pyrethrins

FAO specification 2009:

Pyrethrin (I+II) = 71%

Cinerin (I+II) = 21%

Jasmolin (I+II) = 7%

Studied W.S.P.E. provided by

Pyrethrum Board of Kenya.


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“Pyrethrins” Standards ?

Analyte Chem Service

(GC-FID Cert.)

Botanical Resources Australia ##

(PBK Mercury Reduction method)

Pyrethrins I 57.3% 59.6%

Pyrethrins II 42.7% 40.4%

% Pyrethrins 52.0% 20.53%

All values normalized to 100% Pyrethrins for comparison purpose.

## Botanical Resources Australia (BRL 06-1148, Batch 0106-521-6102)., reported the Pyrethrins I and II by

mercury reduction method.

- Chem Service Cat No N-13151

Pyrethrins I = Pyrethrin 1 + Jasmolin 1 + Cinerin 1

Pyrethrins II = Pyrethrin 2 + Jasmolin 2 + Cinerin 2

--- Lowest MRL : 0.05mg/kg for plant / animal matrices

--- Seems challenging to use these as reference standard for

trace residue analysis in food.


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“Pyrethrins” standard commercial availability ?

Analyte W.S.P.E. 1995-1999 # (Multiple techniques -

Aventis Environmental

Sciences )

USP standard

(LC/DAD

Cert.)

Sigma-

Aldrich

(LC/DAD

Cert.)

Dr.

Ehrenstorfer

(LC/DAD

Cert.)

Botanical

Resources

Australia ##

(PBK method)

Cinerin I Avg: 10.0% (7.6-18.0%) 5.8% 5.6% 4.7% 6.7%

Jasmolin I Avg: 5.3% (4.4-6.1%) 3.6% 3.7% 4.7% 3.4%

Pyrethrin I Avg: 42.4%(37.7-50.0%) 49.1% 50.1% 48.8% 50.5%

Cinerin II Avg: 8.8% (6.7-10.7%) 5.4% 5.3% 4.7% 5.1%

Jasmolin II Avg: 4.8% (2.7-5.8%) 2.6% 2.7% 2.3% 2.6%

Pyrethrin II Avg: 28.7%(18.7-34.0%) 33.6% 32.5% 34.9% 31.7%

% Pyrethrins Avg: 51.4%(48.0-56.4%) 50.3% 52.2% 43% 20.53%

All values normalized to 100% Pyrethrins for comparison purpose.

# Recalculated from Table VI cited in Journal of Chromatographic Science, Vol. 39, November 2001

## Botanical Resources Australia (BRL 06-1148, Batch 0106-521-6102)., reported the Pyrethrins I and II by mercury reduction

method. Individual anlayte ratio estimated from our In-house LC-MS/MS Study.

- USP Certificate on Pyrethrum Extract (Cat. 1585505) / Sigma-Aldrich (Cat No 33739) / - Dr. Ehrenstorfer (Cat No

16620000)


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Meptyldinocap (MRL item 209)

Residue definition:

“Sum of meptyldinocap and 2,4-DNOP, expressed as

meptyldinocap”

What is “Dinocap’ ?

What is “Meptyldinocap” ?

What is “2,4-DNOP” ?



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Pesticides and other agrochemicals — Common names

[ISO 1750:1981]

IUPAC name:

“An isomeric reaction mixture of

2,6-dinitro-4-octylphenyl crotonates and

2,4-dinitro-6-octylphenyl crotonates”

Total six isomers from Dinocap (excluding E/Z form)

Crotonate = E-isomer

2,6- or 2,4- dinitrophenyl substituted ” (~1:2 isomer ratio)

“Octylphenyl” (means ~1:1:1 isomer ratio of)

“methyl n-heptyl-”

and “ethyl n-hexyl-”

and “n-propyl n-pentyl-” isomers (JMPR report 1998)

What is “Dinocap” ?


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AMENDMENT 6: Meptyldinocap [ISO 1750:1981 / DAM 6]

IUPAC name:

“mixture of 75–100% (RS)-2-(1-methylheptyl)-4,6-

dinitrophenyl crotonate and 25–0% (RS)-2-(1-methylheptyl)-

4,6-dinitrophenyl isocrotonate”

CAS Registry Number: 6119-92-2


Amended the IUPAC name, molecular structure, CAS RN

previously listed in page 63, ISO 1750:1981 / AMD.5:2008


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AMENDMENT 6: Meptyldinocap [ISO 1750:1981 / DAM 6]


(75–100% E-isomer) (25–0% Z-isomer)

(RS)-mixture (RS)-mixture

2-(1-(methylheptyl) 2-(1-(methylheptyl))


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2,4-DNOP

= 2-(1-methylheptyl)-4,6-dinitro-phenol

= Meptyldinocap-phenol

CAS RN: 3867-22-7

What is “2,4-DNOP” ?

See also:

1) http://www.lgcstandards.com/

2) “Analysis of Dinocap and Meptyldinocap by modified QuEChERS” (EPRW 2016)

accessible from website: http://www.eurl-pesticides.eu/


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Carbendazim (MRL item 49)

Residue definition:

“Sum of benomyl, carbendazim and thiophanate-methyl, expressed

as carbendazim” [Same as CODEX definition, 34th CCPR (2002)]

What is “Benomyl” ?

What is “Carbendazim” ?

What is “Thiophanate-methyl” ?

What are their relations ?

How to analyze Carbendazim against residue definition?


Pesticides and Definition

related Compounds

CAS-RN Structure

Carbendazim 10605-21-7

Benomyl 17804-35-2

Thiophanate-methyl 23564-05-8


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Fate of Benomyl in acidic, neutral or alkaline medium

Large excess of butyl-isocyanate drives equilibrium to benomyl side. References:

1) Figure 1, Benomyl (JMPR 1973); JMPR evaluation 1998

2) Pages 14-16, CIPAC handbook-D

Acidic / neutral

Carbendazim

+ Butyl-isocyanate

(“BIC” a severe lachrymator)

Alkaline

“STB” (3-butyl-1,3,5-

triazino[1,2a]benzimidazole-

2,4(1H,3H)-dione)

Benomyl

Analytical aspects :

JMPR report 1999: Carbendazim definition covers the use of

carbendazim or from the use of benomyl or thiophanate-methyl

JMPR refers analysis of benomyl to “carbendazim monographs”

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In acidic extraction conditions:

• Benomyl determined as carbendazim

• Carbendazim

• Thiophanate methyl

Study the spike recovery of benomyl determined as

carbendazim

Residue definition:

“Sum of benomyl, carbendazim and thiophanate-methyl,

expressed as carbendazim”


Trends on International Analytical Performance Criteria

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EU and CODEX guideline documents SANTE - EU CCPR - CODEX

Jan 2012 –

SANCO/12495/2011

Jan 2014 –

SANCO/12571/2013

Jan 2016 –

SANTE/11945/2015

April 2016 – REP16/PR

– Appendix XI (Step 5) Draft

April 2015 – CX/PR 15/47/10

– Appendix 1

(Step 4 before meeting, back to Steps 2-4 after

meeting) Draft ×

May 2014 – REP14/PR

– Appendix XI1 (Step 3) Draft

May 2013 – REP13/PR

–Revocation of “Analysis of Pesticide Methods –

Recommended Methods” (CODEX STAN 229-1993)

Start the drafting of “Criteria specific for methods”


Trends on International Analytical Performance Criteria

Updated performance criteria such as:

Retention time / relative retention time criteria

Identification

Confirmation

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Please refer to the guideline documents for details.


Optimization of Pesticide Residue Methods

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Sample preparation related considerations:

Residue definition

Homogenization method of samples

Sample weight, sample wetting

Choice of extraction salt / buffer *

Extraction solvent *

pH of extraction solvent vs pKa / pH of analyte *

Extraction temperature

Extraction time and extraction device *

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* For example: Carbosulfan / Carbofuran / Benfuracarb

can be analysed under same extraction run.



Analysis-related considerations:

Dilution factor, dilution solvent

Salt content of LC mobile phase

LC tubing / mixer configurations

Pre-concentration of sample extract

GC-column selection

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Thank You

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