49145602

• FINAL REPORT

TITLE
Independent Laboratory Validation (ILV) of the
BAYER Method TD-002-W13-01: The Detennination of Thiodicarb and
its Metabolite Methomyl in Water using LC/MS/MS

TEST GUIDELINES
US EPA Test Guidelines:
OCSPP 850.6100, OPPTS 850.7100 and OCSPP 860.1340(c) (6)

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 Study Number: METDL004 Final Report Page 10 of 94

2.0 INTRODUCTION

This report describes the independent laboratory validation (IL V) of Bayer Analytical
Method TD-002-Wl3-0l, "An Analytical Method for the Determination of Residues of
Thiodicarb and its Metabolite Methomyl in Water Using LC/MS/MS" performed by ADPEN
•
Laboratories, Inc. The analytical method is presented in Appendix 1.

This study was designed to satisfy harmonized guideline requirements described in US EPA
Test Guidelines OCSPP 850.6100: Environmental Chemistry Methods and Associated
Independent Laboratory Validation (Reference 2), OPPTS 850.7100 (Reference 3), and
OPPTS (OCSPP) 860.1340(c) (6) (Reference 4). This study was conducted in compliance
with EPA FIFRA Good Laboratory Practice Standards, 40 CFR Part 160 (Reference 5).

3.0 MATERIALS AND METHODS

3.1 Reference Substances

The following reference substances were obtained from Bayer CropScience:

Standard name: Thiodicarb

IUPACname: dimethyl-N,N-[thiobis[(methylimino)carbonyloxy]]bis[ethanimidothioate]
CAS number: 59669-26-0

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Empirical formula: C10H1sN4O4S3 Molecular structure:
Molecular Weight: 354.47 g/mol
Standard Number: K-1470
Ref. Substance Lot: 0428200301
GLP purity: 99.8%
Expiration date: 10-Feb-2019
Storage conditions: Refrigerator (E-109)
~-----------------~
Standard name: Methomyl
IUPACname: methyl-N-[[(methylamino)carbonyl]oxy]ethanimidothioate
CAS number: 16752-77-5 (E: 19929-35-9; Z: 19928-37-1)
Empirical formula: CsH10N2O2S
Molecular structure:
Molecular Weight: 162.21 g/mol 0

_..,.....sYN' ~
Standard Number: K-2059
Ref. Substance Lot: 1218200306
• • t1r,\> purity:· ~
_..,.....cH3
99.5% H3C 0 N
••••
• miration date: 11-Feb-2018
H

: ~tage conditions: Refrigerator (E-109) CH3

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 Study Number: METDL004 Final Report Page 11 of 94

• 3.2 Test System

The test system for this study was an in-house bulk control surface water sample, which was

The sample was logged into the study through the Laboratory Information Management
System (LIMS) and assigned a unique laboratory code, which is cross-referenced to the
sample number on raw data and detailed residue reports. During the course of this study, the
sample was stored in freezer E-16, which operates at a temperature of less than -20 °C.

Sample extracts from the analytical set for thiodicarb (WO-13052401) were injected
immediately after extraction. Sample extracts from the analytical set for methomyl
(WO-13052404) were stored for one day in freezer E-51 while awaiting LC/MS/MS analysis.
The temperature for freezer E-51 at this time was approximately-20 °C.

3.3 Analytical Procedures

Analytical Method TD-002-Wl 3-01 was independently validated as written. The apparatus
and reagents were used as outlined in the analytical method, see Appendix 1, with equivalent
apparatus or reagents substituted as necessary .

• 3.3.1 Fortifications

Untreated control water samples were fortified using the appropriate fortification standard at
LOQ (0.1 ppb) and lOx LOQ (1.0 ppb) concentrations as per the method. Fortifications used
in this method validation are below.

Fortification Fortification Final

Sample Wt.
Matrix Volume Concentration Concentration Replicates
(mL)
(mL) (ng/mL) (ppb)
0.1 0.01 10.0 0.1 5
Water
0.1 0.10 10.0 1.0 5

3.3.2 Extraction Procedure

1. A 10-mL aliquot of water was transferred in to a 50-mL disposable polypropylene

centrifuge tube.
2. SPE manifold with PTFE needles was prepared and Waters Oasis HLB lee Vac RC
60 mg cartridge was attached.
3. The cartridges were conditioned with 2-mL of methanol (MeOH) followed by of ·
HPLC-grade Water (2 mL). The sample was eluted by gravity.

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4. Recovery samples were fortified using individual standard solutions .
Study Number: METDL004 Final Report Page 12 of 94

5. Samples were capped and briefly shaken to mix and centrifuged at 4000 RPM for
10 minutes.
6. A 5-mL aliquot from Step 5 was loaded on HLB cartridge and was eluted by gravity.
7. Approximately 20" Hg of vacuum was applied for 15 minutes and eluate was
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collected.
8. A 15-mL centrifuge tube was placed under each cartridge.
9. Sample was eluted by gravity with MeOH (2 mL) and the column was dried by
applying approximately 5" Hg vacuum for 1 minute followed by MeOH/DCM
solution (3:1, 1 mL v/v) eluting by gravity.
10. Step 9 was repeated.
11. Samples were evaporated to dryness in N-Evap at 45 °C, approximately 20 psi.
12. Samples were reconstituted with 2.5 mL L-Cysteine HCl in MeOH and vortexed for
20 seconds.
13. Samples were transferred to auto-sample vials for LC/MS/MS analysis.

3.3.3 Modifications

Other than instrument parameter optimizations, the following modification was made to the
analytical procedure.

1. After fortification, recovery samples were centrifuged at the speed of 4,000 rpm

3.3.4
instead of 12,500 gas stated in the method.

Suggestions
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1. Due to criticality of the evaporation step in the procedure, one must consider stopping
the evaporation in nitrogen evaporator a little earlier and use the technique of rolling
the concentration tube and allowing the final volume of solvent to evaporate on its
own.
2. In section 4.1 of Bayer Method TD-002-Wl3-0l, specify the time for which the
primary stock solutions of thiodicarb and methomyl are stable.
3. In section 4.3 of Bayer Method TD-002-W13-0l, clarification is required that two
untreated water samples will be fortified with each analyte separately.

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 Study Number: METDL004 Final Report Page 13 of 94

• 3.4 Instrument Operating Parameters

HPLC Instrument:
HPLC Column:
HPLC Conditions
Agilent 1200 SL
Ascentis® Express C18, 2.1 mm x 100 mm, 2.7 µm
Column Temp: 45 °C
Injection Volume: 5 µL
90/10 Water/Methanol with l0mM ammonium
Mobile Phase A:
formate and 120µL/L formic acid
10/90 Water/Methanol with l0mM ammonium
Mobile Phase B:
formate and 120uL/L formic acid
Gradient: Time Flow B
(min) (mL/min) (%)
0 0.5 5
0.5 0.5 5
0.51 0.5 60
1.8 0.5 100
2.5 0.5 100
2.51 0.5 5
5 0.5 5

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MS/MS Conditions
Mass Spectrometer Agilent 6490 Series QQQ
Ion Source Electro Spray Ionization (ESI)
Polarity Positive
Scan Type MRM
Gas Flow 14 L/min
Temperature (TEM) 150 °C
Nebulizer 45 psi
Sheath Gas Heater 300 °C
Sheath Gas Flow 12 L/min
Capillary (V) 3000
V Charging 1500
Thiodicarb Thiodicarb Methomyl Methomyl
MRM Conditions
(Quantitation) (Confirmatory) (Quantitation) (Confirmatory)
Ql m/z 355.06 355.06 163.06 163.06
Q3m/z 88 108 88 105.9
Dwell Time (msec) 100 100 500 300
Frag (V) 380 380 380 380
Collision Energy (CE) 6 10 2 6

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Study Number: METDI..;004 Final Report Page 14 of 94

3.5 Data Acquisition

Peak integration and peak area count quantitation were performed by MassHunter
Quantitative Analysis (version B.04.01) data handling software. A best-fit, linear regression
equation was derived and used in conjunction with the analyte response in each sample to
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calculate the concentration of the analyte. The square of correlation coefficients (R2) for the
calibration curves for each analytical set was greater than 0.99.

Statistical treatment of the data including the calculation of percent recovery, means, and
standard deviations were calculated within LIMS and/or Microsoft® Office Excel spread
sheets. Example calculations are presented in Appendix 4.

4.0 RESULTS AND DISCUSSION

4.1 Method Establishment/Pre-Validation Evaluation

Prior to conducting the IL V, method control was established. Instrument parameters for the
mass spectrometer detector were optimized by infusing standard solutions of the target
analytes. The optimized instrument parameters, analyte retention times, instrument detection
limits, and linearity were established by injecting a series of calibration standards

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 Study Number: METDL004 Final Report Page 15 of 94

4.5 Protocol Changes

Protocol change number 1 was issued to update the experimental design section to instruct
the fortification of control water samples should be performed separately in two validation
sets due to the rapid degradation of thiodicarb to methomyl.

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5.0 CONCLUSIONS

In summary, ADPEN Laboratories successfully and independently validated Bayer

Analytical Method TD-002-W13-01 on the second trial.

The method was demonstrated to be suitable for the determination of thiodicarb and
methomyl in water at an LOQ of 0.1 ppb and at 1Ox LOQ. The method is well-written and
contains sufficient information to guide the analyst through the procedure for the first time .

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Study Number: METDL004 Final Report Page 1:S:of94

TABLE 1. Flow Diagram of the Analytical Procedure

Transfer 10mL of sample water to a 50-mL conical centrifuge tube

+
Prepare SPE manifold with PTFE needles, and attach labeled SPE cartridges
•
+
Condition cartridges with 2mL of MeOH by gravity flow
+
Condition cartridges with 2mL ofHPLC Water by gravity flow
+
Fortify recovery samples as required, using individual standard solutions
(Cap and briefly shake to mix)
+
Centrifuge samples @ 4,000 rpm for 10 minute
+
Run 5-mL aliquot of sample through cartridge by gravity flow
+
Dry cartridge by applying vacuum at ~20" Hg for 15 minutes

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(discard all eluates)
+
Place a 15-mL centrifuge tube under each cartridge
+
Elute with 2mL MeOH by gravity flow
+
Dry cartridge by applying vacuum at ~5" Hg for 1 minute
+
Elute with lmL MeOH/DCM solution (3/1) and dry cartridge by vacuum
(~5Hg for 1 minute)
+
Repeat above step
+
Dry samples in N-Evap@ 45°C, ~30psi
(for approximately 30 minutes)
+
Reconstitute samples in 2.5mL L-Cysteine HCl in MeOH and vortex for 20 seconds
+
Transfer samples to LC vials, cap vials and samples are ready for analysis

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(Store samples in freezer@< -10 °C if not analyzed immediately)
 Study Number: METDL004 Final Report Page 19 of 94

• TABLE 2. Water Fortification Levels

Lab Code
Fortification
Level
(ppb)
Number of
Samples

Analytical set name: WO-13052401 (thiodicarb)

13052401-MB-1 Method Blank 1
130513004-00IC,
Control 2
130513004-001D
13052401-Recoveryl-1,
13052401-Recoveryl-2,
13052401-Recoveryl-3, 0.1 5
13052401-Recoveryl-4, '
13052401-Recoveryl-5
13052401-Recovery2-1,
13052401-Recovery2-2,
13052401-Recovery2-3, 1.0 5
13052401-Recovery2-4,
13052401-Recovery2-5
Analytical set name: WO-13052404 (methomyl)

• 13052404-MB-1
130513004-00lE,
130513004-00lF
13052404-Recoveryl-l,
13052404-Recoveryl-2,
Method Blank

Control
1

13052404-Recoveryl-3, 0.1 5
13052404-Recoveryl-4,
13052404-Recoveryl-5
13052404-Recovery2-1,
13052404-Recovery2-2,
13052404-Recovery2-3, 1.0 5
13052404-Recovery2-4,
13052404-Recovery2-5