DEPARTMENT for ENVIRONMENT, FOOD and R URAL AFFAIRS CSG 15

Research and Development

Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to resreports@defra.gsi.gov.uk

Project title Development of methods of analysis for pesticide formulations

DEFRA project code PS2507

Contractor organisation Central Science Laboratory

and location
Sand Hutton
York

Total DEFRA project costs £ 62,747.81

Project start date 10/01/03 Project end date 31/03/04

Executive summary (maximum 2 sides A4)

The work reported here covers support for surveys undertaken on behalf of PSD under the non-R&D
Memorandum of Understanding. This has included method development and validation in support of the
pyrethrins, malathion and dimethoate surveys and in support of an enforcement action on products. Surveys'
findings are reported separately to PSD.

The pace of development of active ingredients and formulation types is much faster than the process of
international collaborative method development and hence the need for continuous method development. The
level of development required for each exercise varies depending on applicability of published methodology. In
some cases the published methods require modification and validation before they can be applied, but in other
cases the methods are developed in their entirety.

Participation in collaborative trials is essential to enable the publication of referee methods, thus contributions
to four trials were undertaken during the year.

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 Project Development of methods of analysis for pesticide DEFRA PS2507
title formulations project code

Scientific report (maximum 20 sides A4)

Objective 1 of this project was ' To develop analytical methods for the analysis of the components of pesticide
formulations, including active ingredients, impurities and contaminants that may have significant toxicological
or environmental implications. The methods developed will be utilised for scheduled formulation surveys and
enforcement analyses'.

A full report of the work carried out under this objective will be presented in the report required by milestone
01/03; a synopsis of the work conducted is given below.

Pyrethrins Survey

Although there have been three previous studies concerning pyrethrum insecticides, a problem with the
methodology was encountered which took considerable resource to identify and resolve. The method used
had been tested by joint CIPAC/AOAC trial but has not been published. Along with the measurement of the
pyrethrin content the study required the measurement of the piperonyl butoxide (synergist) content. The
piperonyl butoxide content is measured using the internal standard esbiol with triplicate determinations for
each sample. During analysis of samples it was observed that the precision of the measurement was not
acceptable. It was clear that the source of the error was the internal standard response but as the method had
been used successfully in previous studies the first approach to overcome the problem was to repeat the
preparation of samples and standards (as noted earlier, the method requires that samples are prepared in
triplicate). This did not resolve the problem and although tests showed that the instrument was performing
satisfactorily a thorough examination of the key method stages was made before repeating the preparation of
samples for the third time. Once again the poor repeatability was evident. As had been noted in the initial run,
the response of the internal standard was the source of the problem and it was possible to show that a
satisfactory measurement could be made with the data available from the three runs by using external
calibration. As measurement of the piperonyl butoxide content of the samples was now possible, no further
investigation into the reason for the poor repeatability of response of the esbiol internal standard was
undertaken.

The difficulties encountered were raised at a meeting of PAC-UK and other laboratories reported that on
occasion they had noted similar effects. Discussion of the issue led to the suggestion that matrix effects of the
formulations analysed may be the cause. No further work was done to confirm this as the secondary milestone
relating to pyrethrins had been satisfied.

Malathion Survey

A survey of products containing the insecticide malathion required the determination of malathion, iso-
malathion and malaoxon in emulsifiable concentrate formulations and oil-in-water emulsion formulations.
Although there is a published CIPAC method for the determination of malathion in formulations this involves
the use of chloroform, which is a potential carcinogen and also packed column gas chromatography, a largely
out-moded technique. A number of alternative solvents were assessed and the most effective was found to be
acetone. From a number of capillary gas chromatography columns evaluated, a 30 m x 0.25 mm J&W
Scientific DB-1701 (film thickness 0.25 µm) capillary column was found to be most suitable. Using helium as
carrier gas at a flow rate of 1.0 ml/min with an isothermal column temperature of 180 °C the retention time of
the malathion was 10.5 minutes. A split/splitless injector in split mode was used at a temperature of 210 °C,
the injection volume was 1 µl and the FID detector temperature was 290 °C. The precision of the whole
method was tested by performing measurements on five separate preparations of the same sample and the
precision of the instrument method was tested by comparing the peak heights of five repeat injections of a
single extract. This validation showed that the precision of the method was well within the limits suggested by
the modified Horwitz equation and was therefore accepted as suitable for the measurement of malathion in the
samples that comprised the survey.

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 Project Development of methods of analysis for pesticide DEFRA PS2507
title formulations project code

There are two toxicologically significant impurities present in products containing malathion, namely iso-
malathion and malaoxon. The survey required that these two impurities be measured in the samples but there
were no published methods available to do so. A method using GC-MS (Agilent 5973 MSD) was developed
which enabled the simultaneous determination of the two impurities. Samples were diluted in acetone to give
a nominal concentration of 1000 µg/ml malathion and then chromatographed using a 30 m x 0.25 mm J&W
Scientific DB-1701 (film thickness 0.25 µm) capillary column with helium as carrier gas at a flow rate of
1.0 ml/min. The column oven was initially at a temperature of 60 °C for 1 min then ramped at 10 °C/min to
270 °C and held for 5 min giving a total run time of 27 min. The retention times of the malaoxon and iso-
malathion were 19.3 minutes and 21.1 minutes respectively. A split/splitless injector operated in split mode
was used at a temperature of 210 °C and the injection volume was 1 µl. The detector interface was at a
temperature of 280 °C. External calibration was used for quantification.

Dimethoate Survey

The survey of products containing the insecticide dimethoate required (amongst other analyses) the
determination of two toxicologically significant impurities isodimethoate and omethoate. A method for these
impurites in dimethoate formulations was supplied by Cheminova via PSD. The method supplied involved the
use of HPLC with UV detection with separation on a Phenomenex Sphereclone ODS2 column. The mobile
phase was aqueous phosphate buffer and acetonitrile using a gradient programme. However, when this
method was applied to the samples that comprised this survey it was not possible to obtain adequate
separation of the isodimethoate from interfering peaks in all samples despite the fact that all samples were
emulsifiable concentrates. An alternative method using GC-FID was developed in-house. The method used a
30 m x 0.25 mm J&W Scientific DB-1701 (film thickness 0.25 µm) capillary column with helium as carrier gas
at a flow rate of 1.8 ml/min. The column oven was initially at 100 °C and immediately ramped at 20 °C/min to
200 °C then held for 3 mins and then further ramped at 5 °C/min to 240 °C and held for 2 mins giving a total
run time of 18 mins. The retention times of the isodimethoate and omethoate were 7.5 and 10.5 minutes
respectively. A split/splitless injector in split mode was used at a temperature of 270 °C and the injection
volume was 1 µl the detector was at a temperature of 300 °C. External calibration was using to calculate the
isodimethoate and omethoate content of the samples. This method successfully resolved the peaks of interest
and enabled the measurement of these two impurities.

Enforcement action

Enforcement actions require a rapid response as PSD may have to ask manufacturers to withdraw products
from the market pending the results of analysis, this clearly can have severe implications for the manufacturer
involved. In the case of this action PSD were alerted to presence of a parallel product on the UK market which
potentially failed to meet the specifications of the master product. The parallel product appeared to be
deteriorating during storage and it was therefore suspected that there was a deficiency in the preservative
content. PSD requested a measurement of the preservative content of both the parallel and master products.
The master product was specified as containing the biocide Proxel GXL as preservative (the active ingredient
in this preservative is 1,2-benzisothiazol-3(2H)-one. A literature search did not identify any references for the
analysis of 1,2-benzisothiazol-3(2H)-one in any matrix. An attempt was made to determine the 1,2-
benzisothiazol-3(2H)-one content of the products using HPLC with UV detection. The HPLC column was a
250 x 4.6 mm Zorbax ODS (particle size 5 µm) and the mobile phase was 50 % methanol 50 % water
containing 1 % acetic acid. The flow rate was 1.0 ml/min and the injection volume was 10 µl. The detection
was by UV absorbance with the detection wavelength 225 nm. The retention time of the 1,2-benzisothiazol-
3(2H)-one was 3.5 minutes. Using these experimental conditions the 1,2-benzisothiazol-3(2H)-one was
separated from all the other components in the formulation but the detection limit of the method was
inadequate.

A more sensitive method was developed using LC-MS that enabled a comparison of the two products. The
instrumentation used was a Sciex API2000 (Applied Biosystems) triple quadrupole mass spectrometer with
1100 (Agilent) HPLC system. The mass spectrometer was operated in the positive electrospray mode with
selected reaction monitoring (SRM) of two transitions for 1,2-benzisothiazol-3(2H)-one. The SRM transitions
were m/z 152>134 and m/z 152>109 with a dwell time of 250 ms per transition. The chromatography used a
HyPurity C18 (Thermo) analytical column of dimensions 150 x 2.1 mm (5 µm) and a 4 x 2 mm C18 guard
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cartridge (Phenomenex). Mobile phase A was 10 mM aqueous ammonium acetate, mobile phase B was
methanol. The injection volume was 10 µl and gradient elution commenced at 30 % B, rising linearly to 90 %
B over 6 min, remaining at 90 % B for a further 10 min before returning to the initial conditions. The re-
equilibration time was 5 min and the flow rate was 200 µl/min. A switching valve was used to divert to waste
the first 2.5 min of eluant before switching to the mass spectrometer. After elution of the 1,2-benzisothiazol-
3(2H)-one, the eluant was switched back to waste (at 10.5 min) so that the higher concentration of
propyzamide active ingredient did not reach the ion source and contaminate it.

This method was developed and the samples analysed in a short time limit to meet the requirements of PSD;
ensuring in these types of action there are no unnecessary adverse effects on the manufacturers involved.

CIPAC Collaborative Trials

Objective 2 of this project was 'Participate in collaborative trials organised by PAC-UK and CIPAC, as part of
the validation procedure for proposed new international methods'.

During the period covered by this report, CSL participated in four CIPAC (Collaborative International
Pesticides Analysis Council) and PAC-UK collaborative trials involving the determination of deltamethrin, d-
phenothrin, picloram, prallethrin in technical materials and formulations. All trials were completed within
deadline. The collaborative trial organisers will present the data at the annual CIPAC meeting that will be held
in Brno, Czech Republic in June 2004.

Deltamethrin method trial

The deltamethrin trial was a full CIPAC trial organised by Bayer CropScience that involved an HPLC method
for measuring the deltamethrin, a pyrethroid insecticide, in technical materials, tablets, suspension
concentrates, emulsifiable concentrates and water dispersible granules. The method used external
calibration. Samples were prepared in duplicate in 80 % iso-octane / 20 % 1,4dioxane and each duplicate was
injected twice. The HPLC column was a 250 x 4 mm packed with Nucleosil 100-5-CN (particle size 5 µm) and
the mobile phase was 94 % iso-octane 6 % 1,4 dioxane containing 0.15 % water. The flow rate was 1.5
ml/min and the injection volume was 20 µl. The detection was by UV absorbance with the detection
wavelength 230 nm. The retention time of the deltamethrin was 6.6 minutes. The results were required by the
end of February 2004 and CSL reported its data on 11th of that month.

Picloram method trial

The picloram trial was a PAC-UK pilot trial organized by Dow Agosciences that involved an HPLC method for
the determination of picloram, a pyridinecarboxylic acid herbicide in technical materials and soluble
concentrates. The method used internal calibration. Samples were prepared in triplicate in 60 % water 40 %
acetonitrile containing the internal standard and each preparation was injected twice. The HPLC column was
150 x 4 mm packed with ODS-AQ S (particle size 5 µm) and the mobile phase was 83 % water 2 % acetic acid
15 % acetonitrile. The flow rate was 1.5 ml/min and the injection volume was 10 µl. The detection was by UV
absorbance with the detection wavelength 240 nm. The retention time of the internal standard (benamide)
was 3.7 minutes and of picloram, 6.1 minutes. The results were required by the end of February 2004 and
CSL reported its data on 11th of that month.

d-phenothrin method trial

The d-phenothrin trial was a full CIPAC trial organized by the Sumitomo chemical company. The trial tested a
GC method for the determination of d-phenothrin, a pyrethroid insecticide, in technical materials (two batches)
and the liquid phase of aerosol formulations (three batches). The method used internal calibration. Samples
were prepared in duplicate in acetone containing the internal standard and each preparation was injected
twice. The GC capillary column was 30 m X 0.25 mm coated with 14 % cyanopropylphenyl and 86 % dimethyl
polysiloxane at a film thickness of 0.25 µm. The carrier gas was helium at a velocity of 35 cm/min and oven
temperature was isothermal at 230 oC. The injection was made using a split/splitless injector in split mode at a
temperature of 255 oC and the injection volume was 1 µl. The detector was FID at a temperature of 255 oC.
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 Project Development of methods of analysis for pesticide DEFRA PS2507
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The retention time of the internal standard (m-terphenyl) was 8.7 minutes and of the d-phenothrin was 21.3
minutes. The results were required by the end of January 2004 and CSL reported its data on 19th of that
month.

Prallethrin method trial

The prallethrin trial was a full CIPAC trial organized by the Sumitomo chemical Company. The trial tested a
GC method for the determination of prallethrin, a pyrethroid insecticide, in technical materials (two batches)
and liquid vaporizer (three batches) formulations. The method used internal calibration. Samples were
prepared in duplicate in acetone containing the internal standard and each preparation was injected twice.
The GC column was 30 m X 0.25 mm coated with 14 % cyanopropylphenyl and 86 % dimethyl polysiloxane at
a film thickness of 0.25 µm. The carrier gas was helium at a velocity of 35 cm/min and oven temperature was
isothermal at 245 oC. The injection was made using a split/splitless injector in split mode at a temperature of
270 oC and the injection volume was 1 µl. The detector was FID at a temperature of 270 oC. The retention
time of the internal standard (triphenyl phosphate) was 10.2 minutes and of the prallethrin was 4.7 minutes.
The results were required by the end of January 2004 and CSL reported its data on 19th of that month.

Further details of the methods are provided in Surveillance Reports issued to PSD and will be included in a full
R&D report currently in preparation.

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