mm s r4
US Environmental Protection Agency
Office of Pesticide Programs
ECM for Pyriofenone in Water - MRID
49256133
June, 2017
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Huntingdon Life Sciences
JSM0058
2. Materials
2.1 Analytical Standard 1 - IKF-309
Chemical name:
(5-chIoro-2-methoxy-4-methyl-3-pyridiiiyl)
(2)3,4-trimethoxy-6-methylphenyl)
methanone (CAS)
(5-chloro-2-methoxy-4-methyI-3-pyridyl)(2,3,4-
tr imethoxy-6-methy lpheny l)ketone (IUPAC)
Structural fonnula:
Purity:
Expiry date:
Batch number:
Appearance:
Storage conditions:
99.19%
September 2013
0608
White powder
Frozen (approximately -20°C, in the dark)
A Certificate of Analysis is presented in Appendix 1.
2.2 Control matrices
The surface water sample was obtained from a local source (Costessey Pits No. 1) and the
drinking water from a source within the Environmental Analysis Department. Upon receipt
the water samples were allocated unique Huntingdon Life Sciences, Environmental Analysis
Department identification numbers and stored at approximately +4°C prior to use.
The surface water was characterised in a separate study and is presented in the following
table:
Parameter
Found Value
pH
Dissolved Oxygen
Conductivity
Alkalinity
Total Hardness
Total Organic Caitwn
Dissolved Organic Carbon
8.11
11.11 mgOi/L at I9.9°C
543 (iS/cm at 6,0°C
260 mg/L as CaC03
376 mg/L as CaC03
5.804 mgC/L
5.660 mgC/L
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3. Methods
3.1 Validation
Sub-samples of surface and drinking water were fortified with known concentrations of the
test substance and analysed according to the following regime:
2 sub-samples of untreated sample matrix
5 sub-samples of untreated sample matrix fortified at the LOQ (0,05 pg/L)
5 sub-samples of untreated sample matrix fortified at 0,5 fig/L
These samples were then analysed using the analytical methodology, with each sample
injected onto the chromatographic system once.
3.2 Final extract stability
An experiment was set up to demonstrate the stability of the analyte under the typical storage
conditions of the final extracts if they are not quantified immediately after preparation.
Processed control extracts, fortified with IKF-309 were stored at approximately -20°C in the
dark (i.e. fa a freezer).
Aliquots of each of the control sample extracts were fortified with IKF-309 at a concentration
of 1 ng analyte/mL of final extract. The concentration of analytcs in the stored extracts was
determined at day 0 and after 6 days. The concentration of the analytes in freshly fortified
control extracts was also determined at that time.
3.3 Matrix effects
Any possible sample matrix effects were investigated by the comparison of the instrument
response to the analytes in the fortified final extract samples with the response of the analytes
in solvent based calibration standard solutions prepared at the same time.
3.4 Analytical method
Samples were diluted with acetonitrile, Quantitation was performed using liquid
chromatography with tandem mass spectrometric detection (LC-MS/MS).
The analytical method is presented in Appendix 3.
The standard solutions used in this study were also used in other GLP studies being
performed for the same Sponsor. The use of these standard solutions is fully traceable to the
other studies and copies of the standard solution preparation are included in the raw data
package for these studies.
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A stock standard solution of IKF-309 was prepared by dissolving an accurately weighed
amount of the analyte in a suitable volume of acetonitrile, correcting for purity as appropriate.
The stock solution was fiirther diluted with acetonitrile to produce fortification solutions at
10 ng/mL and 100 ng/mL concentrations.
The instrument calibration solutions, over the concentration range 0.01 ng/mL to 1 ng/mL,
were prepared by serial dilution of the fortification solution in aeetonitrile:water (50:50 v:v),
as detailed below:
Standard
solution used
{ng/mL)
Volume
taken («nL)
Final volume
(mL)
Nominal
concentration
(ng/mL)
100
0.1
10
1
too
0.08
10
0.8
100
0.04
10
0.4
100
0.02
10
0.2
1
1
10
0.1
1
0.8
10
0.08
I
0.4
10
0.04
1
0.2
10
0.02
1
0.1
10
0.01
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3.S Calculation of results for validation samples
Test samples were quantified using the following equation;
Residue found (mg/kg) = xx—x D
M
Where x (residue concentration in final solution) was calculated using the linear regression
y — q
m x + c where * (concentration in ng/mL) = -
m
intercept
slope
peak area of sample
matrix concentration (g/mL)
dilution factor
c
m
y
M
D
Example calculation of IKF-309 detected in drinking water fortified at 0,05 pg/mL
(09/00/9375 F0.05 D, analysis batch 3), The primaiy data for this sample is presented in
Table 8, Appendix 2,
Linear regression y = m x + c
67,745 = 2162,92* + 13.5074
where y = 67,745
m = 2162.92
c= 13.5074
Therefore, concentration of IKF-309 (x) = —13.5074 _ qq2508 ng/mL
2162.92 *
Matrix concentration = 0.5 mL matrix/mL final extract
Dilution factor = 1
IKF-309 detected (jig/L) = — 2508 n^m— = 0.0502 ng/mL = 0,0502 pg/L
0,5 mL/mL
0,0502 pg/Lx 100
Recovery (%) = — = 100%
0,05 pg/L
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Appendix 3 Analytical Method
DETERMINATION OF RESIDUES OF IKF-309 IN WATER
1. General principle
Samples are diluted with acetonitrile. Quantitation is performed using liquid chromatography
with tandem mass spectrometry detection (I.C-MS/MS).
2. Apparatus, glassware etc
Balances (various ranges)
Volumetric flasks (various sizes)
Syringes (various sizes)
Volumetric pipettes (various sizes)
Measuring cylinders (various sizes)
Glass scintillation vials (20 ml.)
3. Materials
Ammonium formate
Acetonitrile
Formic acid
Methanol
Water
4. Preparation of reagents
Preparation of acetonitrile: water (50:50 v:v):
Acetonitrile (250 mL) is added to water (250 mL) and mixed thoroughly.
Preparation of mobile phase A - watc r: me than o I:formic acid (90:10:0.1 v:v:v) containing
0.01 M ammonium formate:
Methanol (100 mL), ammonium formate (0.6 g) and formic acid (1 mL) are added to HPLC
water (900 mL) and mixed thoroughly prior to use.
Preparation of mobile phase B - methanol:formic acid (100:0.1 v;v):
Methanol (1000 mL) is mixed thoroughly with formic acid (1 mL).
Note: variable quantities of the above may be prepared by adjusting the constituent
quantities accordingly.
Typical Grade
AR
HPLC
AR
HPLC
HPLC
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5, Analytical standard solutions
An appropriate amount of the test substance (corrected for purity) is accurately weighed and
dissolved in acetonitrile to give a stock standard solution. Appropriate dilutions of the stock
standard solutions are made with acetonitrile to give fortification standard solutions.
The fortification solutions are progressively diluted with acetonitrile:water (50:50 v:v) to
produce a series of instrument calibration solutions in the range 0.01 to 1 ng/mL.
6. Procedure
6.1 Transfer a sub-sample (10 mL) into a scintillation vial.
6.2 Add fortification solutions at this stage if required.
6.3 Add an aliquot (10 mL) of acetonitrile.
6.4 Perform any further dilutions using acetonitrile:water (50:50 v;v)» as required.
6.5 Quantify the samples by the use of LC-MS/MS.
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7. LC-MS/MS conditions
Instrument:
Ionisadon mode:
Ion monitoring details:
Column:
Mobile phase A:
Mobile phase B:
Gradient:
Injection volume:
Flow rate:
Retention rime:
LOD:
LOQ:
Quattro LC / Varian 1200 / Aequity TQD
Positive electrospray
MRM mfz 366>184 (quantitation)
MRM mfz 366>209 (confirmation)
Phenomenex Luna Cg (15 em x 2 mm)
Methanol: water (10:90 v:v) + 0,01 M ammonium
formate + 0.1% formic acid
Methanol + 0,1% formic acid
Time
A
B
(minutes)
(%)
(%)
0
30
70
6
0
100
10
0
100
11
30
70
15
30
70
20 nl
0.2 mL/min
Approximately 5.5 minutes
0.01 ng/mL (= 0.02 jig/L in sample matrix)
0,05 jig/L
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