mm s r4
US Environmental Protection Agency
Office of Pesticide Programs
Independent Laboratory Validation for
Pyriofenone in Water - MRID 49321801
June, 2017

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II. MATERIALS
A.	Equipment
The equipment that was used is listed below:
-	Balance, Analytical, Mettler Toledo XS204
Volumetric flasks, glass: 10, 50 and 100 mL
Bottles, amber glass with Teflon lined cap: 30, 60, and 120 mL
Clear glass vials: 22 mL
Volumetric glass pipette: various sizes
-	Graduated Cylinders: various volumes
Micropipette, Drummond Wiretrol disposable micropipettes: various volumes
Disposable Pasteur pipettes, glass
-	Repeating Pipette, Eppendorf Stream
-	HPLC vials, clear glass: 1.8 mL
-	AB Sciex API4000 LC-MS/MS with Shimadzu LC-20AD HPLC Pumps,
Shimadzu SCL-10A VP Controller, Shimadzu SIL-20AC Autosampler
B.	Reagents and Standards
The following chemicals were used:
Chemical
Grade
Manufacturer
Distributer
Part No:
Acetonitrile
Optima
Fisher
Fisher
A996-4
Ammonium Formate
(>99%)
Not Given
Alfa Aesar
VWR
AA14517-
30
Formic Acid (88%)
ACS
Fisher
Fisher
A118P-500
Methanol
ChromAR
Macron
VWR
MK304110
Water
HPLC
Macron
VWR
MK679510
Water
HPLC
Fisher
Fisher
W5-4
Preparation of Reagent Solutions:
Acetonitrile: HPLC grade water (50:50, v/v): Prepared by adding 250 mL of
acetonitrile to 250 mL of HPLC grade water and mixing well.
Mobile Phase A:
Methanol: formic acid (100:0.1, v/v): Prepared by adding 1 mL of formic acid to
1000 mL of Methanol and mixing well.
Mobile Phase B:
HPLC grade water: methanol: formic acid (90:10:0.1, v/v/v) containing 0.01 M
ammonium formate: Prepared by dissolving 0.6 g of ammonium formate in 900
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mL of HPLC grade water and adding 100 mL of methanol and 1 mL of formic
acid. Mix well.
1. Reference Substance
The analytical reference standard was received in good condition on June
14, 2012 from Midwest Research Institute, Kansas City, MO. The
certificate of analysis for the standard is in the archives at GPL. The
following table contains detailed information for the analytical standard
used in this study.
Analytical
Standard
CAS#
Batch #
Purity
(%)
Expiration
Date
IKF-309
688046-61-9
0608
99.19
09/23/2018
Upon receipt, the neat IKF-309 standard was stored in a freezer set to
maintain < -10 °C.
2. Preparation of Standard Solutions
The IKF-309 reference substance was used in the preparation of the
fortification and calibration solutions. Preparation and dilution data forms
pertaining to the stock and working solutions are located in the raw data.
a.	Stock Solution
On March 10, 2014, 10.1 mg of IKF-309 reference standard was
weighed directly into a 10-mL volumetric flask and diluted to 10
mL with acetonitrile. After correcting for purity, the stock solution
contained 1.00 mg/mL IKF-309 (Solution A).
b.	Intermediate Solution
A IOO-jliL aliquot of Solution A was diluted to 100 mL with
acetonitrile, resulting in a solution that contained 1.00 |ig/mL IKF-
309 (Solution B). Solution B was used to prepare the 100 ng/mL
fortification solution.
c.	Fortification Solutions
A 5-mL aliquot of Solution B was diluted to 50 mL with
acetonitrile, resulting in a solution that contained 100 ng/mL IKF-
309 (Solution C). Further, a 5-mL aliquot of Solution C was
diluted to 50 mL with acetonitrile, resulting in a 10 ng/mL IKF-
309 solution (Solution D). Aliquots of Solution D were used to
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fortify at the LOQ level. Aliquots of Solution C were used to
fortify at the lOx LOQ level. Solution C was used to prepare
calibration standards. Solutions B, C, and D were given an
expiration of three months. The fortification solutions and
calibration standards were stored frozen (< -10 °C) when not in
use.
d. Calibration Standards
All calibration standards were diluted into acetonitrile: HPLC
grade water (50:50, v/v). The calibration standards were given an
expiration of three months. The calibration standards were
prepared by diluting the solutions as listed in the table as follows
into volumetric flasks:
Initial
Volume of
Final
Final
Standard
Solution
Solution
Volume
Solution
Concentration
ID
(mL)
(mL)
ID
(ng/mL)
C
1
100
E
1.00
C
0.8
100
F
0.800
c
0.4
100
G
0.400
c
0.2
100
H
0.200
E
1
10
I
0.100
E
0.8
10
J
0.0800
E
0.4
10
K
0.0400
E
0.2
10
L
0.0200
E
0.1
10
M
0.0100
C. Safety and Health
Material Safety Data Sheets (MSDS) should be consulted anytime an analyst is to
start work with an unfamiliar chemical. Proper personal protective equipment
must be used during the execution of this method. Avoid breathing chemical
vapor and avoid chemical contact with eyes and skin. MSDS for the chemicals
used in this analysis are located in Appendix C. There are no procedural steps
that require special precautions to avoid safety or health hazards.
III. METHODS
A. Principal of Analytical Method
The analysis of surface and drinking water was performed according to the
reference method titled "IKF-309 Validation of Methodology for the
Determination of Residues in Surface and Drinking Water" (contained in
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Huntingdon Life Sciences Document Number: JSM0058). The limit of
quantitation (LOQ) and limit of detection (LOD) were defined as 0.05 |ag/L (ppb)
and 0.02 (ig/L (ppb) for IKF-309, respectively.
The method validations for both surface water and drinking water were performed
on March 11, 2014. All samples for each validation were extracted in one
analytical set. The two sets consisted of one reagent blank sample (HPLC grade
water), two control samples, five LOQ laboratory fortification samples and five
lOx LOQ laboratory fortification samples. Prior to extraction, a unique laboratory
code designation was assigned by GPL to each sample. The laboratory code
consisted of the last three digits of the GPL study number; the sample set
designation and a sample number (e.g., 531ILV01-1).
Aliquots (10 mL) of control matrix water were fortified. An equal amount (10
mL) of acetonitrile was added to each sample. Samples were vialed and analyzed
by LC-MS/MS.
B. Analytical Procedure
1. Control Matrixes
a.	Drinking Water
The drinking water control matrix was obtained from the
municipal supply at GPL on 03/11/2014. The sample was
collected into a 4-L amber bottle and was kept refrigerated (~4 °C)
until use.
b.	Surface Water
The surface water control matrix was obtained from the Fresno
Irrigation District Canal "Herndon No. 39" at a point near the
Gates Avenue Bridge on 02/25/2014. Sub-portions of this sample
were taken and labeled as "Herndon39-140225" and were then
transported by GPL personnel to BSK Laboratories in Fresno,
California for non-GLP characterization. The non-GLP
characterization results of the sample are presented below:
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Parameter
Found Value
pH
7.3
Dissolved Oxygen
9.7 mg/L
Conductivity
42 (imhos/cm
Alkalinity
17 mg/L as CaC03
Total Hardness
12 mg/L as CaC03
Total Residues
31 mg/L
Total Organic Carbon
1.6 mg/L
Dissolved Organic Carbon
1.8 mg/L
Sub-portions from the sample "Herndon3 9-140225-R"
(refrigerated after collection) were used for the method validation.
2.	Preparation of Samples
Sub-samples (10 mL) of the control water matrixes were measured into
22-mL clear glass vials.
3.	Fortifications
Independent laboratory validation samples were fortified at the LOQ (0.05
fxg/L) or lOx the LOQ (0.5 jag/L). Fortifications were performed using
Wiretrol disposable micropipettes to directly fortify the 10-mL samples as
follows:
Fortification Level
Amount and Concentration of IKF-309
Spiking Solution Used
LOQ (0.05 (xg/L)
50 (J.L 10.0 ng/mL
lOx LOQ (0.5 (xg/L)
50 \iL 100 ng/mL
4. Extraction
After fortification, an aliquot (10 mL) of acetonitrile was added to each
sample. Samples were shaken by hand for approximately 5 seconds.
Samples were vialed and submitted for analysis by LC-MS/MS.
C. Instrumentation
Instrument:	AB Sciex API4000 LC/MS/MS with Shimadzu LC-
20AD HPLC Pumps, Shimadzu SCL-10A VP
Controller, Shimadzu SIL-20AC Autosampler
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HPLC Column: Phenomenex Luna C8
150 x 2.00 mm, 5 (im (100 A)
Part # 00F-4040-B0
Serial #568517-6
Guard Column: NA
Data System: Analyst Chromatography Data System version 1.5.2,
AB Sciex
Mobile Phases:
A)	Methanol: Formic Acid (100: 0.1, v/v)
B)	Methanol: HPLC grade water: formic acid
(10:90:0.1, v/v/v) containing 0.01 M ammonium
formate
Flow Rate:
Run Time:
Injection Volume:
Gradient Program:
Time (minutes)
%A
%B
0.0
70
30
6.0
100
0
10.0
100
0
11.0
70
30
15.0
70
30
0.2 mL/minute
15.0 minutes
20 nL
Column Heater: NA
Retention Time:
IKF-309: 6.2 minutes
Mass Spectrometer Parameters (operated in LC-MS/MS mode):
AB Sciex API-4000 Acquisition Parameters
(ESI interface, MRM mode, Positive,

Unit/Unit Resolution)
IKF-309
Ql
Q3
Dwell
(m/z)
(m/z)
(msec)
Primary
366.0
184.3
500
Confirmatory
366.0
209.3
500
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Parameter
Setting
CUR:
20
GS1:
40
GS2:
40
IS:
4500
TEM:
450
CAD:
10
ihe:
ON
DP:
70
CE:
35
EP:
10
CXP:
10
The instrument parameters were optimized for analyte sensitivity and
resolution prior to the chromatographic run. The exact parameters were
documented with the data set.
D. Potential Interferences
1. Matrix Interference
The detection technique is highly selective for this method. No
interferences arising from co-eluting compounds from either water type
were observed.
2.	Reagent and Solvent Interference
High purity solvents and reagents were used for this assay. No
interferences were observed.
3.	Labware Interference
This method uses mostly disposable labware. No interferences from the
labware use were observed.
E. Confirmatory Techniques
The independent laboratory validation sets were run by LC-MS/MS with
monitoring of two ion transition pairs. As this method is highly selective, no
additional confirmatory technique was used.
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F.	Time Required for Analysis
One hour was required for one person to prepare an analysis set from the time
samples were prepared to LC-MS/MS analysis. Automated LC-MS/MS analysis
was performed overnight. An additional 0.5 hours was spent on data calculation
and tabulation the following day. Due to the analysis time (15 minutes per
sample), at most, two calendar days are needed to prepare an analysis set and to
calculate and tabulate the data.
G.	Modification or Potential Problems
There were no modifications to the method. There were no potential problems
encountered. However, the first analysis of the validation set for surface water
showed an approximate 500% percent recovery of IKF-309 in one LOQ sample.
The results of this analysis set are not reported. It was suspected that an error had
been made during sample vialing. The set of samples and calibration standards
were re-vialed and analyzed. The results of the second analysis set met all
acceptance criteria and have been reported.
H.	Methods of Calculation
Analyst Chromatography Data System version 1.5.2, a product of AB Sciex, was
used to acquire, integrate and calculate the concentrations IKF-309 as ng/mL
using the linear regression function with no weighting. The calibration was not
forced through the origin. For the regression calculations, concentration was
designated as the independent variable and plotted on the x-axis. Peak area
response was designated as the dependent variable and plotted on the y-axis.
From this regression curve, a slope, a correlation coefficient and other parameters
of the standard curve were calculated. Calibration standards were injected every
three to five sample injections as well as at the beginning and end of the injection
sequence. Nine different standard concentrations were injected within the
analytical set. The concentrations (ng/mL) of IKF-309 detected in method
validation sample extracts were interpolated from the standard calibration curve.
The concentration as \igfL of residue found in samples was then calculated with
Microsoft® Excel using the following equation:
jug/L = (ng/mL from curve) x (Final Vol. in mL) x 1 uzx 1000 mL
(Sample amount in mL) x 1000 ngx 1 Liter
Recovery of the analyte from fortified samples was calculated as follows:
% Recovery = (Measured Concentration, us/L) x 100
(Theoretical Concentration, jug/L added)
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An example calculation for drinking water for a IKF-309 laboratory fortification
(primary ion) in set 531ILV02, sample 531ILV02-10 lOx LOQ sample fortified at
0.500 jj.g/L, is as follows:
standard curve equation: y = 3.62 x 10 5 (x) + (-561)
where x = IKF-309 concentration in ng/mL and
y = peak response =93423.0
IKF-309 concentration from the curve =0.260 ng/mL
ue/L = (0.260 ne/mL IKF-309) x (20 ml) x luex 1000 mL = 0.520 fjg/L
(10 mL) x 1000 ngxl Liter
% recovery = 		 X100 = 104%
0.500 fig/L
No detectable residues were measured in any control samples. Laboratory
fortification samples were not corrected for reported control responses. Rounding
differences result in minor variations in values between the results obtained using
the standard curve equation and peak area response above in the calculations
versus those values in the report tables and raw data.
I. Statistical Procedures
Laboratory statistical procedures included calculation of arithmetic mean, the
corresponding standard deviation (where n > 3), coefficient of variation and 95%
confidence interval for analyte recovery data. Linear regression analysis was
applied to LC-MS/MS calibration curves for the determination of slope,
y-intercept and correlation coefficient values.
J. Chromatograms
Example chromatograms are presented in Appendix E of this report.
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Analysis of IKF-309 in Water by LC-MS/MS
Measure a 10 mL sub-sample of water into a 22-mL clear vial
I
Fortify as necessary
Add 10 mL of acetonitrile
Shake by hand for approximately 5 seconds
I
Load samples onto LC-MS/MS
If further dilution is necessary,
dilute with Acetonitrile: HPLC grade water (50:50, v/v)
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