Test Material: Nicosulfuron 

MRID: 48790911 

Title: Analytical method for the determination of nicosulfuron (DPX-V9360), 

IN-V9367 and IN-J0290 in water using HPLC/ESI-MS/MS. 

MRID: 48790913 

Title: 

Independent laboratory validation of DuPont-32132, “Analytical method 

for the determination of nicosulfuron (DPX-V9360), IN-V9367 and INJ0290 in water by HPLC/ESI-MS/MS”. 

EPA PC Code: 129008 

OCSPP Guideline: 850.6100 

For CDM Smith 

 

Primary Reviewer: Lisa Muto Signature: 

 Date: 3/20/14 

Secondary Reviewer: Dan Hunt Signature: 

Date: 3/20/14 

 

QC/QA Manager: Joan Gaidos Signature: 

Date: 3/20/14 
Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV) 

Page 2 of 7 

Analytical method for nicosulfuron in water 

Reports: ECM: EPA MRID No. 48790911. Morgan, E.A. and Cabusas, M.E.Y. 2012. 

Analytical method for the determination of nicosulfuron (DPX-V9360), INV9367 and IN-J0290 in water using HPLC/ESI-MS/MS. Laboratory Project 

ID: DuPont-32132. Report prepared by E.I. du Pont de Nemours and 

Company, Newark, Delaware; sponsored and submitted by E.I. du Pont de 

Nemours and Company, Wilmington, Delaware; 54 pages. Final report 

issued March 16, 2012. 

ILV: EPA MRID No. 48790913. Robaugh, D.A. 2012. Independent 

laboratory validation of DuPont-32132, “Analytical method for the 

determination of nicosulfuron (DPX-V9360), IN-V9367 and IN-J0290 in 

water by HPLC/ESI-MS/MS”. DuPont Study No: DuPont-32578. Pyxant 

Labs Project ID: 2388. Report prepared by Pyxant Labs Inc., Colorado 

Springs, Colorado; sponsored and submitted by E.I. du Pont de Nemours and 

Company, Newark, Delaware; 92 pages. Final report issued March 30, 2012.

Document No.: MRIDs 48790911 & 48790913 

Guideline: 850.6100 

Statements: ECM: The study was conducted in a GLP compliant facility following 

standard operating procedures but not with the restriction of compliance with 

USEPA FIFRA GLP standards, 40 CFR, Part 160 (p. 3). Signed and dated 

Data Confidentiality, No Data Confidentiality, GLP, and Certification of 

Authenticity statements were provided (pp. 2-4). A Quality Assurance 

statement was not provided. 

ILV: The study was conducted in accordance with the USEPA FIFRA GLP 

standards (40 CFR Part 160) and OECD Principles of GLP (p. 3). Signed 

and dated Data Confidentiality, No Data Confidentiality, GLP, Quality 

Assurance and Certification of Authenticity statements were provided (pp. 2-

5). 

Classification: This analytical method is classified as Acceptable. However, the LOD and 

LOQ of nicosulfuron in this method for water may not be low enough to 

resolve nicosulfuron at toxicologically relevant concentrations for aquatic 

plants. There appears to be other methods available with lower LOQs for 

nicosulfuron in water. 

PC Code: 129008 

Reviewer: Gabriel Rothman Signature: 

Environmental Scientist Date: May 5, 2014

All page citations refer to MRID 48790911 (ECM) unless otherwise noted. 

Executive Summary

This analytical method, Laboratory Project ID: DuPont-32132, is designed for the quantitative 

determination of nicosulfuron, IN-V9367 and IN-J0290 in water using LC/MS/MS. The method 

is quantitative for nicosulfuron and its transformation products at the stated LOQ of 0.10 ng/mL. 
Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV) 

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The lowest toxicological level of concern in water was not reported. No major issues were 

discovered by the independent laboratory. 

Table 1. Analytical Method Summary 

Analyte(s) by 

Pesticide

MRID

EPA 

Review Matrix Method Date Registrant Analysis

Limit of 

Quantitation

(LOQ)

Environmental 

Chemistry 

Method

Independent 

Laboratory 

Validation

Nicosulfuron 

(DPX-V9360), 

IN-V9367 and 

IN-J0290 

48790911 48790913 Water 3/16/2012

E.I. du Pont 

de Nemours 

and 

Company 

LC/MS/MS 0.10 ng/mL 

I. Principle of the Method 

Samples (20 g) were acidified to a pH of 3.5 to 4.0 with 1 M formic acid then filtered through an 

Oasis® HLB SPE cartridge (pp. 9, 14, 18). The analytes were eluted with methanol:0.50 M 

ammonium hydroxide (9:1, v:v). The eluent was acidified to a pH of ca. 4.5 with 1 mL of 1 M 

formic acid and reduced to ≤2 mL under a stream of nitrogen at ca. 25-30°C. The volume was 

adjusted to 2 mL with water, if necessary, then to 5 mL with 5 mM ammonium formate:methanol 

(19:1, v:v). The resulting solution was filtered (13-mm, 0.45-μm PTFE filter) prior to analysis. 

Samples were analyzed for nicosulfuron, IN-V9367 and IN-J0290 by reversed-phase HPLC 

(Phenomenex® Luna, 4.6 mm x 150 mm, 3 μm phenyl-hexyl column) using a mobile phase 

gradient of (A) 0.1 mM formic acid in 0.1 mM ammonium formate and (B) methanol [percent 

A:B at 0.00 min. 95:5 (v:v), 4.00 min. 50:50, 4.10 min. 35:65, 7.00 min. 10:90, 7.10-10.00 min. 

5:95, 10.10-12.00 min. 95:5] with MS/MS-ESI+ detection and Multiple Reaction Monitoring 

(MRM; pp. 9, 10, 14, 20). The ratios of two MRM parent-to-daughter ions were monitored 

(quantitative and confirmatory) per analyte. Injection volumes were 10-20 μL; an injection 

volume of 10 μL was indicted for pond water in order to reduce matrix effects. 

The LOQ was the same in the ECM and ILV (0.10 ng/mL; p. 24; p. 11 of MRID 48790913). In 

the ECM, the LOD was estimated to be one-third of the LOQ, ca. 0.03 ng/mL. The LOD was not 

defined in the ILV. 

II. Recovery Findings 

ECM (MRID 48790911): Mean recoveries and relative standard deviations (RSD) were within 

guideline requirements (mean 70-120%; RSD ≤20%) for analysis of nicosulfuron, IN-V9367 and 

IN-J0290 in tap (drinking) water, pond water and well water (p. 24; Table 1, pp. 28-36). 

Confirmation of the identified peaks was based on the relative ratio of its two parent-to-daughter 

ion transitions; all of these ratios were accurate within the range of the equivalent ion ratios of 

the calibration standards (pp. 25-27). 

ILV (MRID 48790913): Mean recoveries and RSDs were within guideline requirements for 

analysis of nicosulfuron, IN-V9367 and IN-J0290 in pond water (p. 18; Tables 4-6, pp. 24-26). 
Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV) 

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Quantitative ion and confirmatory ion results were comparable. The method was validated with 

the second trial (p. 11; see Comment #2). 

Table 2. Initial Validation Method Recoveries for Analytes in Water

Analyte

Fortification 

Level 

(ng/mL)

Number 

of Tests

Recovery 

Range (%) 

Mean 

Recovery (%)

Standard 

Deviation (%) 

Relative Standard 

Deviation (%)

Well Water (Kimblesville, Pennsylvania) 

Nicosufluron 

(DPX-V9360) 

0.10 (LOQ) 5 90-94 92 1.5 1.5 

1.0 5 84-96 91 4.3 4.7 

IN-V9367 0.10 (LOQ) 5 93-104 98 4.7 4.8 

1.0 5 100-112 106 4.7 4.4 

IN-J0290 0.10 (LOQ) 5 86-91 88 2.1 2.4 

1.0 5 89-95 93 2.4 2.6 

Lums Pond Water (Lums Pond, Delaware) 

Nicosufluron 

(DPX-V9360) 

0.10 (LOQ) 5 71-94 82 8.5 10.4 

1.0 5 91-109 98 6.9 7.1 

IN-V9367 0.10 (LOQ) 5 74-92 82 8.4 10.3 

1.0 5 90-112 97 9.3 9.6 

IN-J0290 0.10 (LOQ) 5 79-88 83 3.8 4.6 

1.0 5 71-82 77 5.5 7.1 

Tap (Drinking) Water (Newark, Delaware) 

Nicosufluron 

(DPX-V9360) 

0.10 (LOQ) 5 82-89 85 2.5 2.9 

1.0 5 88-102 95 5.3 5.5 

IN-V9367 0.10 (LOQ) 5 88-94 92 3.1 3.4 

1.0 5 98-110 106 5.2 4.9 

IN-J0290 0.10 (LOQ) 5 68-83 77 6 7 

1.0 5 74-87 83 5.3 6.4 

Data were obtained from p. 24 and Table 1, pp. 28-36 in the study report. 

Table 3. Independent Validation Method Recoveries for Analytes in Pond Water

Analyte

Fortification 

Level 

(ng/mL)

Number 

of Tests

Recovery 

Range (%)

Mean 

Recovery (%)

Standard 

Deviation (%) 

Relative Standard 

Deviation (%)1

Water Recoveries – Quantitative ion 

Nicosufluron 

(DPX-V9360) 

0.10 (LOQ) 5 82-94 89 4.6 5 

1.0 5 84-92 88 3.2 4 

IN-V9367 0.10 (LOQ) 5 85-92 90 3.1 3 

1.0 5 94-99 97 2.2 2 

IN-J0290 0.10 (LOQ) 5 79-95 87 6.0 7 

1.0 5 75-80 78 1.9 2 

Confirmation ion 

Nicosufluron 

(DPX-V9360) 

0.10 (LOQ) 5 86-100 94 5.6 -- 

1.0 5 81-91 86 4.0 -- 

IN-V9367 0.10 (LOQ) 5 90-93 92 1.1 -- 

1.0 5 94-103 99 3.2 -- 

IN-J0290 0.10 (LOQ) 5 72-84 77 4.4 -- 

1.0 5 75-82 77 2.9 -- 

Data were obtained from p. 18; Tables 4-6, pp. 24-26 of MRID 48790913. 
Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV) 

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1 Reviewer-calculated for quantitative ion; confirmation ion raw data not supplied in ILV study report (see DER 

Attachment 2). 

III. Method Characteristics 

The LOQ was the same in the ECM and ILV (0.10 ng/mL). In the ECM, the LOQ was defined as 

the lowest fortification level which obtained average recoveries of 70-110% and a RSD <20% (p. 

24). The LOD was not determined experimentally; however, it was estimated as one-third of the 

LOQ (ca. 0.03 ng/mL). In the ILV, the LOQ was reported from the ECM, and no justification 

was provided (p. 11 of MRID 48790913). The LOD was not defined. 

Table 4. Method Characteristics 

Nicosulfuron IN-V9367 IN-J0290 

Limit of Quantitation (LOQ) 0.1 ng/mL 0.1 ng/mL 0.1 ng/mL 

Limit of Detection (LOD) ca. 0.03 ng/mL ca. 0.03 ng/mL ca. 0.03 ng/mL 

Linearity (calibration curve r2 and 

concentration range) 

r

2

 = 0.99961

(0.25-10 ng/mL) 

r

2

 = 0.9951-0.99991

(0.25-10 ng/mL) 

r

2

 = 0.9929-0.99931

(0.25-10 ng/mL) 

Repeatable Yes Yes Yes 

Reproducible Yes2 Yes2 Yes2

Specific Yes Yes Yes 

Data were obtained from p. 24; Figure 1, p. 37; Appendices 1-2, pp. 46-54. 

1 ILV calibration curves yielded similar linearity, r2 = 0.9974-0.9993, for concentration range of 0.25-10 ng/mL (see 

p. 17 and Figures 1-3, pp. 27-29 of MRID 48790913). 

2 The ILV successfully validated the method at the LOQ in the second trial, after decreasing the injection volume to 

10 μL as specified in the ECM for pond water (see Comment #2). 

IV. Method Deficiencies and Reviewer’s Comments 

1. It may be possible to obtain lower LOCs and LOQs for nicosulfuron in water using other 

methods. The reviewer found references to other LOQs determined for nicosulfuron of 

0.01 ppb, approximately one order of one magnitude lower than this evaluated analytical 

method (Battaglin et al., 2001). 

 

2. In general, toxicologically relevant concentrations of nicosulfuron are > 1.0 ppm range 

for aquatic animals. However, there is uncertainty with the toxicity of aquatic plants as 

this study has not been submitted to the Agency, but has been requested as part of the 

Registration Review program. Given nicosulfuron’s phytotoxic mode of action, it is 

likely that aquatic plants will be far more sensitive to nicosulfuron exposure than aquatic 

animals. Therefore, the analytical method should possess the ability to measure the 

lowest concentrations possible such as indicated by Battaglin et al., 2001 to account for 

nicosulfuron’s potential toxicity to aquatic plants. 

3. In the ILV, Method Validation Trial 1 was unsuccessful (pp. 10, 17-18 and Appendix 3, 

p. 92 of MRID 48790913). After consultation with the Sponsor, the ILV decreased the 

HPLC injection volume to 10 μL, as suggested in the ECM method for pond water (p. 

20), and successfully validated the method with Trial 2. 
Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV) 

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4. The calibration standards in the ECM and ILV included only samples in the range of 0.25 

ng/mL to 10 ng/mL when the LOQ for method validation was a 0.10 ng/mL spiking 

level; however, the linear regressions of the peak area versus concentration of the 

calibration standards were not used to calculate the analyte found (p. 22). The calibration 

standards were used to calculate Response Factors (concentration of standard/peak area 

counts) which were used to calculate the analyte found. Also, the peak areas of the 

samples were within range of the peak areas of the calibration standards, most likely due 

to the concentration procedures used during the method (Appendices 1-2, pp. 46-54). 

5. Matrix characterization of the pond, well and drinking water in the ECM (p. 17) and pond 

water in the ILV was reported (Appendix 1, p. 84 of MRID 48790913). 

6. The linear regression equations of the reviewer-generated calibration curves did not 

exactly match those reported in the ECM and ILV; however, the equations and r2

 values 

were similar, in general. 

7. The reviewer was unable to verify the recoveries for the ECM from data and equations 

provided in Appendices 1-2, pp. 46-54 of the study report. The equation [% recovery = 

(analyte found *100)/(fortification level)] was provided by the study authors; however, 

all of the significant figures of the “analyte found” data were not reported. For example, 

in the recoveries of IN-V9367 in the well water samples dosed at the LOQ, the test 

samples LOQ 1 and LOQ 3 had the same “analyte found” (0.10 ng/mL) and sample 

weight (20.00 g), but differing percent recoveries of 93% and 95%, respectively 

(Appendix 2, p. 53). The reviewer did not calculate “analyte found” from the raw data. 

8. It was reported for the ILV that a single analyst completed a sample set consisting of 15 

samples in ca. 8-10 hours or 2 calendar days (p. 19 of MRID 48790913). 

V. References 

Battaglin W.A., Furlong E.T., and M.R. Burkhardt., 2001. Concentration of Selected 

Sulfonylruear Sulfonamide, and Imidazolinone Herbicides, Other Pesticides, and 

Nutrients in 71 Streams, 5 Reservoir Outflows, and 25 Wells in the Midwestern United 

States, 1998. U.S. Geological Survey, Denver, CO. 

U.S. Environmental Protection Agency. 2012. Ecological Effects Test Guidelines, OCSPP 

850.6100, Environmental Chemistry Methods and Associated Independent Laboratory 

Validation. Office of Chemical Safety and Pollution Prevention, Washington, DC. EPA 

712-C-001. 

40 CFR Part 136. Appendix B. Definition and Procedure for the Determination of the Method 

Detection Limit-Revision 1.11, pp. 317-319. 
Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV) 

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Attachment 1: Chemical Names and Structures 

Nicosulfuron; DPX-V9360 

IUPAC Name: 1-(4,6-Dimethoxypyrimidin-2-yl)-3-(3-dimethylcarbamoyl-2-

pyridylsulfonyl)urea. 

2-[(4,6-Dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl]-N,Ndimethylnicotinamide. 

CAS Name: 2-[[[[(4,6-Dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-

N,N-dimethyl-3-pyridinecarboxamide monohydrate. 

CAS Number: 111991-09-4. 

SMILES String: c1(c(nccc1)S(=O)(=O)NC(=O)Nc2nc(cc(n2)OC)OC)C(=O)N(C)C 

IN-V9367

IUPAC Name: Not reported. 

CAS Name: 2-(Aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide. 

CAS Number: 112006-75-4. 

SMILES String: c1(cccnc1S(=O)(=O)N)C(=O)N(C)C 

IN-J0290 

IUPAC Name: Not reported. 

CAS Name: 4,6-Dimethoxy-2-pyrimidinamine. 

CAS Number: 36315-01-2. 

SMILES String: Nc1nc(cc(n1)OC)OC