2.0 INTRODUCTION
Described in this report is the independent laboratory validation of Syngenta Analytical
Method GRM060.08A (Reference 1) as performed by PASC.
This study was designed to satisfy guideline requirements described in EPA 850.6100 (2012)
(Reference 2). This study was conducted in compliance with EPA FIFRA Good Laboratory
Practice Standards, 40 CFRPart 160.
The residue analytical method is deemed suitable for the determination of Flumetralin in soil.
Soil sample contents are extracted with methanol:water (80/20 v/v). An aliquot of sample is
partitoned into hexane:toluene (50/50 v/v) and submitted to negative-ion chemical ionization
mass spectrometry (GC-NICI-MS) for analysis.
The validated limit of quantitation of method GRM060.08A is 0.01 mg/kg in soil.
3.0 MATERIALS AND METHODS
3.1 Test/Reference Substance
The test/reference substance was obtained from Syngenta Crop Protection, LLC. The
following test/reference substance was used:
Compound Structure
F f,
F'\/ it
xo"
O"
C.-"
Syngenta Code:
CGA41065
Common Name:
Flumetralin
CAS Name:
N-ethyl-N-(2-chloro-6-florobenzyl)-4-trifluromethyl-2,6-
dinitroaniline
Batch ID:
410533
Molecular Weight:
421.7 g/mol
Storage Conditions:
Refrigerate < 30°C
Purity:
99.9%±0.5%
Expiration Date:
End of March 2022
Characterization data for the test/reference standard are maintained by Syngenta Crop
Protection, LLC. The Certificate of Analysis is included in Appendix 2.
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The test/reference substance (Flumetralin) used in this study was procured from Syngenta
Crop Protection, LLC located at the Greensboro facility. All solutions made from
Flumetralin standard were stored according to Section 2 of the method.
3.2 Test System
The test system evaluated for this ILV was Soil.
3.3 Equipment and Reagents
The equipment and reagents used for the ILV were as outlined in the method. Identical or
equivalent equipment and materials were used, as permitted by the method. All solvents and
other reagents must be of high purity, e. g. glass distilled/HPLC grade solvents and analytical
grade reagents.
3.4 Preparation of Standard Solutions
Standard solutions were prepared and stored as recommended in Section 2 of the method
(Reference 1).
3.4.1 Stock Standard
One 100 |j,g/mL stock solution for flumetralin was prepared in acetone.
3.4.2 Fortification Standard
Sample fortification solutions containing flumetralin were prepared by serial dilution in
acetone from the stock solution. The following solutions were prepared: 10.0 ng/mL, 1.0
Hg/mL and 0.1 |j,g/mL for fortification purposes.
3.4.3 Calibration Standard
Calibration standards were prepared by serially diluting stock standards using hexane:toluene
(50/50 v/v). Using equivalent GC-MS instrumentation described in the method, the following
concentration range of standards (0.25 pg/|iL, 0.5 pg/|iL, 10 pg/mL, 2.5 pg/|iL, 5 pg/|iL, and
10 pg/|iL) were prepared and used to construct the calibration plots.
3.5 Analytical Procedures and Modifications
Analytical Method GRM060.08A (Reference 1) was successfully validated by an independent
laboratory as written using the procedures and instrumentation recommended by the method.
Soil contents are extracted with methanol:water (80/20 v/v). An aliquot of sample is
partitioned into hexane:toluene (50/50 v/v) and submitted to negative-ion chemical ionization
mass spectrometry (GC-NICI-MS) for m/z 421, 423, and 391 for analysis. The limit of
quantitation of Analytical Method GRM060.08A (Reference 1) is 0.01 mg/kg in soil.
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3.5.1 Modifications
Syngenta Analytical Method GRM060.08A (Reference 1) was followed as written.
3.5.2 Fortifications
Untreated control soil samples were fortified using 100 |j,L of known amounts of flumetralin
to LOQ and 10X LOQ concentration levels as per the method. See Table 2 for detailed
fortification levels. Fortifications used in this ILV are as follows:
Matrix
Fortification
Fortification
Replicates
Volume (|jL)
Cone. (|ag/mL)
LOQ
100
1
5
10X LOQ
100
10
5
3.5.3 Method Summary
As per Analytical Method GRM060.08A, soil contents are extracted with methanol:water
(80/20 v/v). An aliquot of sample is partitioned into hexane:toluene (50/50 v/v) and
submitted to negative-ion chemical ionization mass spectrometry (GC-NICI-MS) for m/z
421, 423, and 391 for analysis.
3.5.4 Limit of Detection and Limit of Quantitation
The limit of detection (LOD) of the method is defined as the lowest analyte amount injected
on column detectable above the mean amplitude of the background noise at the
corresponding retention time. An estimate of the LOD can be taken as three times
background noise. The limit of detection using the instrumentation for this validation was
estimated to be 0.5 pg on column. Note that the LOD may vary between runs and from
instrument to instrument.
The LOQ of the method is defined as the lowest analyte concentration in a sample at which
the methodology has been successfully validated with a mean recovery of 70 - 110% and a
relative standard deviation of < 20% has been obtained. A limit of quantitation (LOQ) of
0.01 mg/kg in soil was successfully validated in this study.
3.5.5 Detector Linearity
The linearity of the detector response was assessed using a calibration curve generated with
each analysis sequence injected. It was shown that the GC-MS detector response for
flumetralin has a correlation coefficient > 0.995 in the range from 0.25 pg/uL to 10.0 pg/uL
or 0.5 pg to 20.0 pg on column when using a 2 |iL injection volume.
Representative plots of the detector responses versus the analyte concentration for all
calibration points are presented in the Figures Section.
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3.6 Data Acquisition
Peak integration and peak area count quantitation were performed by "Chemstation Software
version G1732BA, B.02.00.589". A best-fit, linear regression equation was derived and used
in conjunction with the analyte response in each sample to calculate the concentration of the
analyte. The square of the correlation coefficients (R ) for the calibration curves for each
analytical set was > 0.995. Recovery results were computed for each sample.
A statistical treatment of the data includes the calculation of averages, standard deviations,
and relative standard deviations. Mean percent recoveries, standard deviations, and relative
standard deviations were calculated using Microsoft Office Excel (2007).
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AITI M HX 3 GC-MS Tuning Procedure
( of Instt uini'tit
"line inMrtimeni mils! he muss calibrated on a regular basis. Perform instrument auto tune of
compound specific tunc using specific calibration mass®,
luniint Ijistruim-nt for ilumctralin
Determine ionization mode and detection (EI or CI).
Perform scan of expected maxscs. Determine target ion and qualifier ions. Target plus two
qualifiers above 100 anin are recommended.
I'or llumetralin. in negative ion chemical ionization mode, the deprotonated molecular ion
generated is selected (in - 421) as the target ion. 'Hie two most sensitive qualifier ions (in r
423 and m r 3
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AITI MHX 4 Method How Chart
Weigh 10 g of soil into a 150 nil, plastic (Nalgene)
4
Add 100 ml methanol: watct (XU 2d \ \ I and shake 1 hour
4
Centrifuge (3500 rpm)
4
Perform !J J* with sail u\tm; 15 ml. extract and 5 nil. toluene: hexanc(50:50 v/v)
4
Centrifuge (3500 rpm)
^4
Dilute 1 miL supernatant (organic phase) to a Html volume of 4 ml using toluene: hexane
(50-50 v v)
4
Transfer i ml, to autosampler vial and analyse by OC-NIC1-MSD
Report Number: GRM060.Q8A Page 3? of 3?
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