US Environmental Protection Agency
Office of Pesticide Programs
Independent Laboratory Validation
for Pyriofenone in Soil - MRID
49256127
June, 2017

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Report/Independent Laboratory Validation (ILV) of IKF-309 in Soil
Document Number: 027336-1
Schedule of Events
Study Initiation Date:	March 17, 2011
Experimental Start Date:	March 24, 2011
Experimental Termination Date: March 30, 2011
Retention of Da ta
Upon completion of the study, the complete study file including all original raw data was
submitted to the Ricerca Biosciences, LLC Corporate Archives for storage.
Conduct of the Study
The study was conducted at the Ricerca Biosciences, LLC AgChem Product Development
Department Laboratories according to the Ricerca Biosciences, LLC protocol "Independent
Laboratory Validation (ILV) of the Residue Analytical Method for Detection of IKF-309 in
Soil (RCC Study #B18843)," Document Number 027336-0.
INTRODUCTION
IKF-309 is under development as an agricultural crop protection agent. As part of the
registration, scientists at RCC, Ltd. developed an analytical method for detection of IKF-309 in
agricultural soil. This report describes the Independent Laboratory Validation (ILV) of this
method.
OBJECTIVE/PURPOSE
The purpose of this study was to perform an independent laboratory validation of the method of
analysis for detection of IKF-309 in agricultural soil.
TEST SUBSTANCE
The sponsor supplied the test substance, IKF-309. The certificate of analysis is included in
Appendix A.
The standard and the stock solutions made from them were stored under freezer conditions
(~ -20 °C). Solubility and stability data for the reference substances, expiration and storage
conditions were the responsibility of the Sponsor. Information concerning the test substance
including purity is provided below:

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HpSftft
Report/Independent Laboratory Validation (ILV) of IKF-309 in Soil
Document Number: 027336-1
• IKF-309
ch3
Common Name:
Chemical Name:
CAS No.:
Molecular Formula:
Batch Number:
Purity:
Expiration Date:
Storage:
ch3
h3c ch3
ch3
IKF-309
(5-Chloro-2-methoxy-4-methyl-3-pyridinyl)
(2,3,4-trimethoxy-6-methylphenyl)methanone (CA)
688046-61-9
Ci8H20C1NO5
0608
99.19%
09/2013 (per Sponsor)
Frozen (~ -20°C)
TEST SYSTEM
The test system (North Dakota Soil IB-2010-JLW-004-01-030) was supplied by the Sponsor and
stored at ~ -20 0°C (in a freezer) until analysis. The soil parameters are summarized in the table
below. The certificate of analysis is included in Appendix A.
Parameters

Percent Sand
67
Percent Silt
15
Percent Clay
18
USDA Textural Class (Hydrometer method)
Sandy loam
Bulk Density (disturbed) gm/cc
1.16
Cation exchange capacity (meq/lOOg)
18.6
% Moisture at 1/3 Bar
24.4
% Moisture at 15 Bar
12.3
% Organic Matter -Walkley Black
2.5
pH in 1:1 soil:water ratio
7.3
Base Saturation Data

Cation
Percent
ppm
Calcium
69.5
2587
Magnesium
15.9
355
Sodium
0.4
17
Potassium
3.0
220
Hydrogen
11.2
21
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Report/Independent Laboratory Validation (ILV) of IKF-309 in Soil
Document Number: 027336-1
The water content of the soil was determined prior to the study. Three aliquots of the wet soil
were weighed into aluminum weighing dishes and dried over the weekend at approximately
100 °C after which the soil samples were re-weighed and the soil moisture determined to be
21.9%. The residues in treated samples were measured on dry soil weight basis. The calculation
used for initial weight for analysis corresponding to 10 g dry weight is shown below:
X = (a x c)/b
X = weight of wet soil corresponding to 10 g dry soil
a = weight of dry soil for analysis (10 g)
b = weight of dry soil after drying
c = initial weight of weight soil for moisture determination (10 g)
Therefore, X = (10 x 10)/7.81 = 12.8 g
ANALYTICAL METHODOLOGY
Extraction
Samples were extracted according to the method provided by the Sponsor:
1.	Approximately 12.8 g of soil samples were weighed into 50-mL polypropylene
centrifuge tubes and fortified.
2.	37.2 mL of acetonitrile (ACN)/HPLC water/hydrochloric acid (HC1) (800/200/5) was
added to the soil sample to give an extraction volume of 40 mL.
3.	The samples were shaken using a laboratory shaker for 30 minutes.
4.	The samples were centrifuged for 10 minutes at -3000 rpm.
5.	An aliquot of 4 mL of the extract was removed and mixed with 20 mL of HPLC water.
SPE Cartridge Clean-up
1.	A Waters Oasis HLB VAC (60 mg) SPE cartridge was placed on an SPE vacuum
manifold and washed with 5 mL methanol followed by 5 mL HPLC water.
2.	The cartridge was loaded with the sample from step # 5 and the cartridge was sucked
with vacuum to dryness. The eluent was discarded.
3.	IKF-309 was eluted with 5 mL of acetonitrile. The fraction was collected into a 10-mL
volumetric flask and the cartridge was sucked with vacuum to dryness.
4.	The volumetric flask was filled to the mark with HPLC water and the sample was
mixed.

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Report/Independent Laboratory Validation (ILV) of IKF-309 in Soil
Document Number: 027336-1
5. Samples fortified with 0.001 mg/kg IKF-309 and control soil samples were directly
analyzed by LC-MS/MS without dilution. Samples fortified with 0.1 mg/kg IKF-309
were diluted 20x using acetonitrile/HPLC water (1:1) before LC-MS/MS analysis.
LC-MS/MS Analysis
Separation of the analyte from soil matrix was achieved by high performance liquid
chromatography (HPLC). Quantitative LC-MS/MS analysis of IKF-309 in the samples
utilized a highly specific and sensitive MRM (Multiple Reaction Monitoring) method. IKF-
309 precursor ion (m/z 366.00) was monitored in Q1 and the fragment ions were monitored
in Q3 (m/z 184.30, primary method and m/z 209.30, confirmatory method). The analyte was
identified by the coincidence of its retention time with the calibrant standards, and
quantitated by integration of the peak area relative to the calibration curve.
The following are the LC-MS/MS parameters used.
HPLC: Two Shimadzu LC20-AD pumps and a Shimadzu SIL-HTA Controller/Autosampler
Column: Inertsil ODS-3, 50 mm x 2.1 mm x 3|i particle size
Injection Volume: 10 |iL
Solvent System:
Solvent A = HPLC water/acetonitrile/formic acid (95:5:0.1%)
Solvent B = HPLC water/acetonitrile/formic acid (5:95:0.1%)
Solvent Program:
Time (minutes)
Flow Rate
(mL/min)
%A
%B
0.00
0.30
50
50
2.00
0.30
0
100
2.50
0.30
0
100
2.60
0.30
50
50
4.50
0.30
50
50
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Report/Independent Laboratory Validation (ILV) of IKF-309 in Soil
Document Number: 027336-1
Mass Spectrometer: SCIEX API 4000
The mass spectrometer was calibrated every four weeks per SOP 04-C023-03.
Mass Spectrometer settings:
Scan Type:
MRM
Polarity:
Positive
Ion Source:
Turbo Spray
Resolution Q1
Unit
Resolution Q3
Unit
Ion Source Gas 1 (GS1):
20.0 psi
Ion Source Gas 2 (GS2):
25.0 psi
Curtain Gas (CUR):
25.0 psi
Collision Gas (CAD):
6.0 psi
IonSpray Voltage (IS):
3500 V
Temperature (TEM):
550 °C
Declustering Potential (DP):
75.0 V
Entrance Potential (EP):
10.0 V
Collision Gas Exit Potential (CXP):
17.0 V
Period 1 settings:
Q1 Mass
(amu)
Q3 Mass
(amu)
Dwell Time
(msec)
Collision
Energy (CE)
366.00
184.30
200.00
32 V
366.00
209.30
200.00
37 V
Retention Time: -3.9 minutes
Methods of Calcula tion
Recoveries
The recoveries of IKF-309 from fortified samples were calculated relative to the linearity
curve generated with each set:
Linear regression formula from calibration curve y = mx + b
ng/mL IKF-309 = ^
m
Where y = Sample peak area
b = Calibration intercept
m = Calibration slope
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Report/Independent Laboratory Validation (ILV) of IKF-309 in Soil
Document Number: 027336-1
„	• , , T \ Sample peak area - intercept
Sample Concentration (ng/mL) =	-—-	—
Slope
ppm IKF-309 =
Sample Cone. (ng/mL) x Final Vol. (mL) x Initial Extract Vol. (mL) x 0.001 //g/ng
Aliquot vol. (mL) x 10 grams
where |ig/g is equivalent to mg/kg and ppm.
Percent Recovery =
Cone, of IKF - 309 Fortified Sample (ppm) - Cone, of Control (ppm)
	x 100
IKF - 309 Fortification Level (ppm)
An example calculation for the recovery of IKF-309 (0.001 ppm fortification) from soil
(sample no. 027336-Soil 0.001 mg/kg A) is shown below:
The calibration curve equation was y = 43400x + (-467) (r = 0.9993)
ng/mL IKF-309 = 3620 ~(~467) = 0.0942 ng/mL*
43400
slight differences from reported data due to rounded numbers are used for calculation.
Calculation of the ppm IKF-309 in soil was calculated as shown:
0.0942 ng/mL x 10 mLx 40 mLx 0.001
ppm IKF-309 =	-	= 0.000942 ppm
4 mLx 10 grams
The percent recovery of IKF-309 equals 0.000942 ppm minus control (0 ppm) IKF-309,
divided by the 0.001 ppm fortification times 100 equals the percent recovery for this
sample at 94.2%.
n n	0.000942 ppm - 0 ppm	^
Percent Recovery =	—	—— x 100 = 94.2%
0.001 ppm
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Report/Independent Laboratory Validation (ILV) of IKF-309 in Soil
Document Number: 027336-1
SIGNIFICANT OBSERVATION
PTFE syringe filters (Fisher Scientific, 0.45 |im, 13 mm, lot #ROMA82407) removed a portion
of IKF-309 from the samples, that is, 15-33% of the 0.001 ppm samples and 12-22% of the 0.1
ppm samples after 20x dilution (primary method, product ion: m/z = 184.3). Therefore, to
analyze soil samples following the RCC method B18843, it may be necessary to remove the
syringe filtration step from the method unless quantitative recovery of IKF-309 through syringe
filters can be demonstrated.
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