Test Material: Nicosulfuron 

MRID: 48790912 

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

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

MRID: 48790914 

Title: 

Independent laboratory validation of DuPont-33143, “Analytical method 

for the determination of nicosulfuron (DPX-V9360), IN-V9367 and INJ0290 in soil using 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 48790912 (ECM)/ 48790914 (ILV) 

Page 2 of 8 

Analytical method for nicosulfuron in soil 

Reports: ECM: EPA MRID No. 48790912. Cabusas, M.E.Y. 2012. Analytical method 

for the determination of nicosulfuron (DPX-V9360), IN-V9367 and INJ0290 in soil using HPLC/ESI-MS/MS. Laboratory Project ID: DuPont33143. 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; 50 pages. Final report issued March 16, 

2012. 

ILV: EPA MRID No. 48790914. Rogers, P. 2012. Independent laboratory 

validation of DuPont-33143, “Analytical method for the determination of 

nicosulfuron (DPX-V9360), IN-V9367 and IN-J0290 in soil using 

HPLC/ESI-MS/MS”. DuPont Project ID: DuPont-33031. Alliance Pharma 

Project No.: 120103. Report prepared by Alliance Pharma, Inc., Malvern, 

Pennsylvania, sponsored and submitted by E.I. du Pont de Nemours and 

Company, Wilmington, Delaware; 52 pages. Final report issued March 22, 

2012. 

Document No.: MRIDs 48790912 & 48790914 

Guideline: 850.6100 

Statements: ECM: The study was not conducted with the restriction of compliance with 

USEPA FIFRA GLP standards, 40 CFR, Part 160, or OECD GLP (p. 3). 

Signed and dated Data Confidentiality, No Data Confidentiality, and GLP 

statements were provided (pp. 2-3). Certification of Authenticity and Quality 

Assurance statements were not provided. 

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

Laboratory Practice (GLP) standards (40 CFR Part 160) and OECD 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 soil were not low enough to resolve 

nicosulfuron at toxicologically relevant concentrations for plants. 

PC Code: 129008 

Reviewer: Gabriel Rothman Signature: 

Environmental Scientist Date: May 5, 2014

All page citations refer to MRID 48790912 (ECM) unless otherwise noted. 

Executive Summary

This analytical method, Laboratory Project ID: DuPont-33143, is designed for the quantitative 

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

quantitative for nicosulfuron and its transformation products at the stated LOQ of 1.0 μg/kg. The 

lowest toxicological level of concern in soil was not reported. No major issues were discovered 

by the independent laboratory. 
Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV) 

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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 

48790912 48790914 Soil 3/16/2012

E.I. du Pont 

de Nemours 

and 

Company 

LC/MS/MS 1.0 μg/kg 

I. Principle of the Method 

Samples (10 g) were extracted twice by shaking for 15-20 minutes with 0.1 M ammonium 

carbonate:acetone (9:1, v:v) then centrifuged (pp. 8, 10, 12-13, 17-19). The supernatant was 

decanted and filtered. One-fourth of the combined extract was purified by passing through an 

Isolute® NH2 solid-phase extraction (SPE) cartridge. The acetone was removed from the 

partially-purified extract by evaporation using nitrogen-evaporator (N2-vap) system at 30°C. The 

reduced extract was acidified to pH 3 with 1M formic acid then purified by passing through 

stacked OasisTM HLB and ENV+® SPE cartridges. The analytes were eluted with methanol:1 M 

ammonium hydroxide (9:1, v:v) solution and methanol. The eluent was reduced to ca. 3 mL in 

an N2-vap system at 30°C. The final volume was adjusted to 5 mL with 5 mM ammonium 

formate:methanol (19:1, v:v). The resulting solution was filtered (25-mm, 0.45-μm PTFE filter) 

prior to analysis. 

Samples were acidified (10 μL of 1M formic acid added to 990 μL of sample) then 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 0.01 mM ammonium formate and (B) methanol [percent A:B at 0.0 min. 95.0:5.0 (v:v), 10-13 

min. 5.0:95.0, 13.1-17 min. 95.0:5.0] with MS/MS-ESI+ detection and Multiple Reaction 

Monitoring (MRM; pp. 8, 10, 13, 19-21). The ratios of two MRM parent-to-daughter ions were 

monitored (quantitative and confirmatory) per analyte. Injection volumes were 75 μL. 

The LOQ was the same in the ECM and ILV (1.0 μg/kg; p. 24; p. 11 of MRID 48790914). In the 

ECM, the LOD was not experimentally determined (p. 24); however, was reported as one-third 

of the LOQ or 0.33 μg/kg for the Drummer clay loam soil (Appendix 4, pp. 47-49). In the ILV, 

the LOD was estimated to be 0.3 μg/kg (p. 11 of MRID 48790914). 

II. Recovery Findings 

ECM (MRID 48790912): 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 clay loam, loam, silty loam and sandy loam soils (p. 23; Table 1, pp. 28-39). 

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. 25-26). 
Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV) 

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ILV (MRID 48790914): Mean recoveries and RSDs were within guideline requirements for 

analysis of nicosulfuron, IN-V9367 and IN-J0290 in one soil (pp. 18-19; Tables 1-6, pp. 22-27). 

Quantitative ion and confirmatory ion results were comparable. The relative ratios of the two 

parent-to-daughter ion transitions were similar between the LOQ and 10xLOQ sample sets 

(Tables 7-9, pp. 28-30). The method was validated with the first trial (p. 10). 

Table 2. Initial Validation Method Recoveries for Analytes in Soil

Analyte

Fortification 

Level 

(μg/kg)

Number 

of Tests

Recovery 

Range (%) 

Mean 

Recovery (%)

Standard 

Deviation (%) 

Relative Standard 

Deviation (%)

Clay loam (Drummer #7 from Illinois) 

Nicosufluron 

(DPX-V9360) 

1.0 (LOQ) 5 99-115 104 6.1 5.8 

10.0 5 95-106 101 4.3 4.2 

IN-V9367 1.0 (LOQ) 5 85-100 90 7.1 7.9 

10.0 5 82-93 88 4.8 5.5 

IN-J0290 1.0 (LOQ) 5 70-85 77 5.7 7.4 

10.0 5 71-78 75 3.0 4.0 

Loam (Mattapex #25 from Maryland) 

Nicosufluron 

(DPX-V9360) 

1.0 (LOQ) 5 94-107 100 5.8 5.8 

10.0 5 96-110 101 5.9 5.9 

IN-V9367 1.0 (LOQ) 5 78-94 87 6.5 7.6 

10.0 5 82-94 87 5.1 5.9 

IN-J0290 1.0 (LOQ) 5 72-89 80 6.4 8.0 

10.0 5 76-85 81 3.8 4.8 

Silty loam (Nambsheim from France) 

Nicosufluron 

(DPX-V9360) 

1.0 (LOQ) 5 92-104 99 4.5 4.6 

10.0 5 96-105 99 3.7 3.7 

IN-V9367 1.0 (LOQ) 5 85-105 97 7.8 8.0 

10.0 5 98-100 98 0.9 0.9 

IN-J0290 1.0 (LOQ) 5 73-86 82 5.5 6.7 

10.0 5 83-85 84 1.0 1.2 

Sandy loam (Speyer 2.2 from Germany) 

Nicosufluron 

(DPX-V9360) 

1.0 (LOQ) 5 94-113 106 8.3 7.8 

10.0 5 97-120 104 9.1 8.7 

IN-V9367 1.0 (LOQ) 5 82-94 89 4.8 5.3 

10.0 5 82-96 88 5.8 6.6 

IN-J0290 1.0 (LOQ) 5 78-92 88 5.8 6.6 

10.0 5 74-95 82 8.1 9.9 

Data were obtained from p. 23; Table 1, pp. 28-39; and Appendix 4, pp. 47-49 in the study report. 
Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV) 

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Table 3. Independent Validation Method Recoveries for Analytes in Soil

Analyte

Fortification 

Level 

(μg/kg)

Number 

of Tests

Recovery 

Range (%)

Mean 

Recovery (%)

Standard 

Deviation (%) 

Relative Standard 

Deviation (%)

Soil Recoveries – Quantitative ion 

Nicosufluron 

(DPX-V9360) 

1.0 (LOQ) 5 105-114 109 3.8 3 

10.0 5 97-108 103 4.8 5 

IN-V9367 1.0 (LOQ) 5 72-84 77 5.4 7 

10.0 5 74-82 78 3.1 4 

IN-J0290 1.0 (LOQ) 5 84-91 89 3.1 4 

10.0 5 72-83 80 4.5 6 

Confirmation ion 

Nicosufluron 

(DPX-V9360) 

1.0 (LOQ) 5 100-120 106 8.8 8 

10.0 5 93-102 98 4.2 4 

IN-V9367 1.0 (LOQ) 5 58-86 71 10.1 14 

10.0 5 70-80 75 3.7 5 

IN-J0290 1.0 (LOQ) 5 82-84 83 0.8 1 

10.0 5 77-88 83 4.2 5 

Data were obtained from pp. 11, 19; and Tables 1-6, pp. 22-27 of MRID 48790914. 

III. Method Characteristics 

The LOQ was the same in the ECM and ILV (1.0 μg/kg). 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 reported as one-third of the 

LOQ (0.33 ng/g) for the Drummer clay loam soil (Appendix 4, pp. 47-49). In the ILV, the LOQ 

was reported from the ECM, and no justification was provided (p. 11 of MRID 48790914). The 

LOD was estimated to be 0.3 μg/kg; no justification was provided. 

Table 4. Method Characteristics 

Nicosulfuron IN-V9367 IN-J0290 

Limit of Quantitation (LOQ) 1.0 μg/kg 1.0 μg/kg 1.0 μg/kg 

Limit of Detection (LOD) 0.33 μg/kg1 0.33 μg/kg1 0.33 μg/kg1

Linearity (calibration curve r2 and 

concentration range) 

r

2

 = 1.00002

(0.25-20 ng/mL) 

r

2

 = 0.99962

(0.25-20 ng/mL) 

r

2

 = 0.99992

(0.25-20 ng/mL) 

Repeatable Yes Yes Yes 

Reproducible Yes Yes Yes 

Specific Yes Yes Yes 

Data were obtained from pp. 22, 24; Figure 1, p. 40; and Appendix 4, pp. 47-79. 

1 Reported for Drummer clay loam soil. The LOD was reported as 0.3 μg/kg in the ILV (p. 11 of MRID 48790914). 

2 ILV calibration curves were linear, r2

 = 0.9940-0.9952, for concentration range of 0.25-20 ng/mL (see p. 19 and 

Figure 4, pp. 40-42 of MRID 48790914). 

IV. Method Deficiencies and Reviewer’s Comments 
Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV) 

Page 6 of 8 

1. The LOD and LOQ arrived at with the analytical method used is not sufficient to address 

the concern for nicosulfuron’s potential to adversely impact plants at very low exposure 

levels. The EC25 determined for the most sensitive endpoint for plant effects is very low 

at 0.00045 lbs./A (seedling emergence endpoint for mustard seeds, MRID No. 

42220001). This endpoint can be triggered with a concentration of < 0.1 ppb of 

nicosulfuron in soil. The method evaluated established the LOD an order of magnitude 

higher at 0.33 ppb and LOQ two order of magnitude higher at 1.0 ppb. While these are 

low concentrations, an analytical method for nicosulfuron extractions from soil should be 

precise enough to resolve nicosulfuron residues at toxicologically relevant concentrations 

for plants. 

2. The reviewer is aware of one other previously developed analytical method for 

nicosulfuron. This method has demonstrated the ability to measure nicosulfuron and 

other sulfonylurea pesticide residues in soils at very low concentrations. This analytical 

method utilizing LC-MS possessed LOQs as low as 6 ppt in soil (Marek and Koskinen, 

1997), approximately 2 orders of magnitude lower than this reviewed analytical method. 

3. In the ECM, the extraction efficiency of the method was evaluated in an aged soil (clay 

loam from Japan) fortified at 50 μg/kg (pp. 13, 16, 26; Appendix 5, p. 50). The original 

method was modified to eliminate the purification and concentration with SPE. 

Recoveries in the aged soil were 94-99%, 93-96% and 89-90% of the applied for 

nicosulfuron, IN-V0367 and IN-J0290, respectively. These recoveries were normalized 

using fresh soil recoveries; normalized recoveries were 101%, 97% and 102% for 

nicosulfuron, IN-V0367 and IN-J0290, respectively. 

4. A reagent blank was not included in the ILV (p. 20; Tables 1-6, pp. 22-27 of MRID 

48790914). 

5. The linear regression equations of the reviewer-generated calibration curves matched 

those reported in the ECM; however, the linear regression equations of the reviewergenerated calibration curves indicated greater linearity than those reported in the ILV. 

6. The reviewer was unable to verify the recoveries for the ECM from data provided in 

Tables 1-6, pp. 28-39 of the study report. The equation [% recovery = (analyte found 

*100)/(fortification level)] was provided by the study authors on pp. 20-21; however, the 

study author noted that only two significant figures of the “analyte found” data were 

reported. For example, in the recoveries of nicosulfuron in the clay loam soil samples 

dosed at the LOQ, the test samples LOQ A and LOQ B had the same “analyte found” of 

1.0 μg/kg, but differing percent recoveries of 101% and 103%, respectively (Table 1, p. 

28). The reviewer did not calculate “analyte found” from the raw data; raw data was only 

provided for one soil, the clay loam. 

7. It was reported for the ILV that a single analyst completed two sample sets consisting of 

6 samples per set in 1.5 working days (p. 20 of MRID 48790914). 
Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV) 

Page 7 of 8 

8. For the ILV, a Phenomenex® Luna Phenyl-Hexyl 3.0 x 150 mm, 3-μm column was used 

at a flow rate of 0.425 mL/min, instead of a Phenomenex® Luna Phenyl-Hexyl 4.6 x 150 

mm, 3-μm column at a flow rate of 1.0 mL/min as described in the ECM (MRID 

48790914, pp. 14-15). The study author stated that the study monitor approved of the 

substitution and that the substitution was demonstrated to be equivalent to that specified 

in the method. 

9. Characterization of the test soils should have included information regarding organic 

carbon content, cation exchange capacity, and soil taxonomic classification as these 

properties may influence recoveries pesticide residues at different spiking levels in the 

analytical method evaluation. 

V. References 

Marek L.J. and Koskinen W.C., 1997. LC-MS Analysis of Polar Pesticides in Soil. RJ Reynolds 

Industry Publication, Richmond, VA. 

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 48790912 (ECM)/ 48790914 (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