JRF Study Number: 240-2-11-5551 (Final Report) Page 2 of 52 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 

No claim of confidentiality is made for any information contained in this study on the basis of its falling 

within the scope of FIFRA Section 10 (d) (1) (A), (B), or (C). 

Sponsor Tiarco Chemical 

 1300 Tiarco Drive 

 Dalton, GA 30720 

 

Signature ___________________________ 

 Sponsor/Submitter 

Date ____________________________



JRF Study Number: 240-2-11-5551 (Final Report) Page 6 of 52 

CONTENTS

PAGE N° 

TITLE PAGE ........................................................................................................................................... 1 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS ............................................................ 2 

STATEMENT OF GLP COMPLIANCE ................................................................................................ 3 

STATEMENT OF COMPLIANCE WITH GOOD LABORATORY PRACTICE ................................ 4 

STATEMENT OF QUALITY ASSURANCE ........................................................................................ 5 

CONTENTS ............................................................................................................................................ 6 

PERSONNEL INVOLVED IN THE STUDY ........................................................................................ 8 

SUMMARY ............................................................................................................................................. 9 

 Infrared Analysis ............................................................................................................................... 9 

 LC-Mass Analysis ........................................................................................................................... 10 

 NMR Analysis ................................................................................................................................. 10 

 Active Ingredient Content ............................................................................................................... 10 

1. INTRODUCTION ........................................................................................................................... 11 

 1.1 Study Objectives .................................................................................................................... 11 

 1.2 Test Facility and Study Period .............................................................................................. 11 

 1.3 Regulatory Guidelines ........................................................................................................... 11 

 1.4 Archives................................................................................................................................. 11 

2. EXPERIMENTAL PROCEDURE ................................................................................................. 12 

 2.1 Test Item ................................................................................................................................ 12 

 2.2 Reference Standard ................................................................................................................ 12 

 2.3 Equipment/Instruments ......................................................................................................... 13 

 2.4 Solvents, Chemicals and Reagents ........................................................................................ 13 

 2.5 Introduction ........................................................................................................................... 13 

 2.6 Interpretation ......................................................................................................................... 13 

 2.7 Methodology for IR Spectral Analysis .................................................................................. 14

 2.8 Procedure for the Determination of Active Ingredient Content ............................................ 14 

 2.8.1 Preparation of Sodium 3-Nitrobenzene Sulfonate Reference Standard 

 Stock Solution .......................................................................................................... 14

 2.8.2 Preparation of Reference Standard Solutions for Linearity ...................................... 14 

 2.8.3 Preparation of Reference Standard Solution for Active Ingredient Analysis ........... 15 
JRF Study Number: 240-2-11-5551 (Final Report) Page 7 of 52 

CONTENTS (Continued)

PAGE N° 

 2.8.4 Preparation of Sample Solutions .............................................................................. 15 

 2.8.5 Instrumental Parameters ........................................................................................... 15 

 2.9 Procedure for LC-Mass Spectral Analysis ............................................................................ 15 

 2.9.1 Preparation of Sodium 3-nitrobenzenes sulfonate Reference Standard and Test 

 Item Solutions........................................................................................................... 15

 2.9.2 LC Mass Parameters ................................................................................................. 16

 2.10 Procedure for NMR Analysis ................................................................................................ 16 

 2.10.1 Preparation of Sodium 3-nitrobenzenes sulfonate Reference Standard and 

 Test Item Solution .................................................................................................... 16 

3. RESULTS ....................................................................................................................................... 17 

 3.1 Infrared Spectra ..................................................................................................................... 17 

 3.2 Mass Analysis ........................................................................................................................ 17 

 3.3 NMR Analysis ....................................................................................................................... 17 

 3.4 Active Ingredient Content ..................................................................................................... 17 

4. CONCLUSION ............................................................................................................................... 18 

5. REFERENCES ................................................................................................................................ 19 

TABLES 

 1. Linearity Data of Sodium 3-Nitrobenzene Sulfonate Reference Standard ............................ 20 

 2. Calculation of Active Ingredient Content .............................................................................. 21 

APPENDICES 

 1. IR, Mass NMR Spectra and HPLC Chromatograms ............................................................. 22 

 2. Signed Study Plan and Study Plan Amendment .................................................................... 35 

 3. Certificate of Analysis of Sodium 3-Nitrobenzenes Sulfonate Technical Sample ................ 49 

 4. Certificate of Analysis of Sodium 3-Nitrobenzenes Sulfonate Reference Standard ............. 50 

 5. GLP Endorsement of Compliance ......................................................................................... 51 

JRF Study Number: 240-2-11-5551 (Final Report) Page 9 of 52 

SUMMARY

This study was conducted to perform the characterization of sodium 3-nitrobenzene sulfonate. The test 

item was supplied by Tiarco Chemical, Dalton, GA 30720. The method was followed as per the 

guidelines of the SANCO/ 3030/99 rev. 4 AND U.S. EPA OPPTS 830.1800. 

IR and Mass analysis were carried out following the Standard Operating Procedures of Jai Research 

Foundation. 

The IR and NMR spectral data were interpretated using standard references William D. H. and Fleming I. 

(1988) and Robert M. S., Francis X. W (1997). 

The structure of sodium 3-nitrobenzene sulfonate test item was confirmed by comparing with sodium 3-

nitrobenzene sulfonate reference standard using FT-IR based on the presence of the characteristic 

absorption bands due to various functional groups; using LC-MS based on molecular mass and retention 

time and using NMR based on the chemical shift and numbers of proton present in test item and standard. 

The active ingredient analysis was carried out using validated analytical method (JRF Study Number: 

228-2-13-5312) 

Infrared Analysis 

The structures of sodium 3-nitrobenzene sulfonate reference standard and test item were confirmed based 

on the presence of the characteristic absorption bands due to various functional groups by infrared 

analysis as described below: 

Functional groups Reference Standard Sample 

- CH stretching 3106.76, 2936.09 and 2873.42 3102.9, 2935.13 and 2873.42 

- CH bending 1467.56 1468.53 

-C=C stretching in benzene ring 1611.23 1610.27 

-NO2 stretching 1536.99 and 1351.86 1536.02 and 1351.86 

-SO3 H stretching 1190.83, 1051.98 and 647.965 1192.76, 1047.16 and 670.142 

-adjacent H- atom 764.637 764.637 
JRF Study Number: 240-2-11-5551 (Final Report) Page 10 of 52 

 LC-Mass Analysis 

Identification of sodium 3-nitrobenzene sulfonate reference standard and test item using LC-MS, based on 

molecular mass and retention time is summarized below: 

Sample Retention Time (min) Mass (m/z) 

Reference Standard 3.999 202.2 

Test Item 4.018 202.2 

NMR Analysis 

Identification of sodium 3-nitrobenzene sulfonate reference standard and test item using NMR, based on 

chemical shift and numbers of proton present are summarised below: 

Molecular Structure: 

SO3

NO2

H

H

H

H

Na - +

1

2

3 4

Identification No of 

Protons Multiplicity 

Chemical Shift (δ) Integration 

Standard Sample Standard Sample 

1 1 Triplet 7.724 – 7.764 7.723 – 7.764 1.09 1.09 

2 1 Doublet 8.216 –8.242 8.216 –8.242 1.06 1.06 

3 1 Doublet 8.327- 8.369 8.327- 8.369 1.11 1.18 

4 1 Singlet 8.666 – 8.675 8.665 – 8.675 1.00 1.00 

Active Ingredient Content 

Active Ingredient Content of sodium 3-nitrobenzene sulfonate was carried out using validate analytical 

method [Reference JRF Study Number: 228-2-13-5312]. 

The mean active Ingredient Content of sodium 3-nitrobenzene sulfonate was 96.88%w/w 
JRF Study Number: 240-2-11-5551 (Final Report) Page 11 of 52 

1. INTRODUCTION 

1.1 Study Objectives 

This study was conducted to perform the characterization of sodium 3-nitrobenzene sulfonate 

(CAS # 127-68-4, US EPA HPV 3). The study was conducted in compliance with the OECD 

Principles of GLP (1998). 

1.2 Test Facility and Study Period

This study was performed at the Department of Chemistry, Jai Research Foundation, Valvada – 

396 108, Dist. Valsad, Gujarat, India. 

Study Initiation : October 12, 2012 

Experiment Start : October 15, 2012 

Experiment Completion : October 15, 2012 

Study Completion : December 05, 2012 

1.3 Regulatory Guidelines 

European Commission: Directorate General Health and Consumer Protection 2000: SANCO/ 

3030/99 rev. 4 “Technical and Material Preparations: Guidance for generating and reporting 

methods of analysis in support of pre- and post-registration data requirements for Annex II (Part 

A Section 4) and Annex III (part A, Section 5) of Directive 91/414/EEC” as amended by 

European Commission Directive 94/37/EC and 96/46/EC. 

U.S. EPA, 1996: OPPTS 830.1800 “Enforcement of Analytical Method” (EPA 712-C-96-013). 

United States Environmental Protection Agency, Product Properties Test Guidelines (August 

1996). 

1.4 Archives 

All original raw data including any storage medium for electronically recorded data, 

documentation, IR spectra, mass spectra, NMR spectra, chromatograms, the signed study plan, 

the study plan amendment, the draft report, a copy of final report and the representative sample of 

the test item will be retained in the GLP Archives at Jai Research Foundation for a period of ten 

years. At the end of this period, the sponsor's instructions will be sought to either extend the 

archiving period or return the archived material to the Sponsor or for the material to be disposed 

of. 
JRF Study Number: 240-2-11-5551 (Final Report) Page 12 of 52 

SO3

NO2

Na

- +

2. EXPERIMENTAL PROCEDURE

2.1 Test Item 

Details of the test item provided by the Sponsor are: 

Test Item Name 

IUPAC Name 

C.A. Name 

CAS N° 

Batch N° 

Analysed Purity 

Manufacture by 

Supplied by 

Date of Manufacture 

Date of Expiry 

Appearance 

Sodium 3-Nitrobenzene Sulfonate 

Benzenesulfonic acid, 3-nitro, sodium salt (1:1) 

Benzenesulfonic acid, 3-nitro, sodium salt (1:1) 

127-68-4 

50623 

96.9% (Refer Certificate of Analysis in Appendix 3) 

Tiarco Chemical 

Tiarco Chemical 

September 08, 2011 

September 08, 2013 

Yellow to tan granular 

Storage Condition 

(at JRF)

As per the instruction received from the Sponsor on storage of the test 

substance, the test item was stored in its original container as supplied by 

the Sponsor at room temperature in the Test Item Control Office (TICO). 

The stability of the test item in storage was the responsibility of the 

Sponsor. 

2.2 Reference Standard 

Reference Standard 

IUPAC Name 

C.A. Name 

Molecular Weight 

Molecular Formula 

CAS N° 

Lot N° 

Date of Receipt 

Date of Expiry 

Purity 

Source 

JRF Entry Number 

Storage Condition 

Sodium 3-Nitrobenzenes Sulfonate 

Benzenesulfonic acid, 3-nitro-, sodium salt (1:1) 

Benzenesulfonic acid, 3-nitro-, sodium salt (1:1)

225.15 

O2NC6H4SO3Na 

[127-68-4]

07315KNV 

August 07, 2012 

August 06, 2017 (as per JRF SOP-152) 

98.0% [Refer Appendix 4] 

Sigma-Aldrich, USA 

CPC-54 

Room Temperature 

Source: Molecular structure was obtained from Internet. 
JRF Study Number: 240-2-11-5551 (Final Report) Page 13 of 52 

2.3 Equipment/Instruments 

Sr. N° Name Model Manufacturer 

1 Balance GR-202 Adair Dutt 

2 FT-IR Spectrometer FT IR-4100 Jasco 

3 LC-MS Agilent 1100 series, binary pump 

with Agilent VWD/MSD Agilent 

4 NMR 300 MHz Bruker 

5 HPLC LC 2010 CHT Simadzu 

2.4 Solvents, Chemicals and Reagents 

Sr. N° Name Grade Supplier 

1 Water Milli- Q Milli pore Milli Q water 

purification system 

2 Acetonirile HPLC s.d fine chem. ltd 

3 Potassium bromide (KBr) IR Merck 

4 Formic Acid HPLC Merck 

5 Deutorated methanol NMR Qualigens 

2.5 Introduction 

Sodium 3-nitrobenzenes sulfonate reference standard and test item were analyzed by FT-IR 

Spectrometer, LC-MSD, NMR and HPLC (for A.I Content). The identification with FT-IR based 

on the presence of the characteristic absorption bands due to various functional groups; using LCMS based on molecular mass and retention time and using NMR based on the chemical shift and 

numbers of proton present in test item and standard. The active ingredient analysis was carried 

out using validated analytical method using HPLC. 

2.6 Interpretation 

The spectral data was interpreted using the standard references viz., William D. H. and Fleming I. 

(1988) and Robert M. Silverstein, Francis X. Webster, 1997. 
JRF Study Number: 240-2-11-5551 (Final Report) Page 14 of 52 

2.7 Methodology for IR Spectral Analysis 

Approximately 2.0 mg of reference standard and test item was mixed with 100 mg (approx.) 

dried potassium bromide separately. The mixture was finely ground using pestle and mortar and 

the mixture was placed in DRA sample holder of sample compartment. Background spectrum 

was recorded before analyzing sample by using blank KBr. 

All samples and background spectra were recorded using the following parameters: 

Instrument : FT-IR Spectrometer 

Wave Number Range : 400-4000 cm-1 

Resolution : 4.0 cm-1 

2.8 Procedure for the Determination of Active Ingredient Content 

2.8.1 Preparation of Sodium 3-Nitrobenzene Sulfonate Reference Standard Stock Solution 

Weight 

(mg) of 

Reference 

Standard 

Final Volume 

(mL) 

Volume made 

using Purity (%) 

Obtained 

Concentration 

(mg/L) 

Identification of 

Reference 

Standard Stock 

Solution 

25.47 25 Milli-Q Water 98.0 998.42 A 

2.8.2 Preparation of Reference Standard Solutions for Linearity 

Reference 

Standard 

Stock Solution 

Solution Taken 

(mL) 

Final Volume 

(mL) 

Volume made 

using 

Obtained 

Concentration 

(mg/L) 

Identification of 

Reference Standard 

Solution 

A 

0.25 

10 Milli-Q Water

24.96 B 

0.50 49.92 C 

0.75 74.88 D 

1.00 99.84 E 

1.25 124.8 F 

1.50 149.76 G 

The reference standard solutions [B, C, D, E, F and G] were injected onto HPLC in duplicate 

using the parameters in accordance with section 2.8.5 and the mean peak area was plotted against 

concentrations (mg/L). The correlation coefficient (r), intercept with y-axis (a) and slope of the 

line (b) were calculated and the regression equation Y = bX + a was established. 
JRF Study Number: 240-2-11-5551 (Final Report) Page 15 of 52 

2.8.3 Preparation of Reference Standard Solution for Active Ingredient Analysis 

The reference standard solution (E), concentration 99.84 mg/L, prepared for linearity was 

used for determination of active ingredient content in the sample. 

2.8.4 Preparation of Sample Solutions 

Replication 

Weight of 

Standard/Test 

Item (mg)

Volume 

Made using 

(mL) 

Identification of 

Stock Solution 

Volume 

taken 

Final Volume 

(mL) 

I 24.20 25 R1 1.0 10 

II 24.34 25 R2 1.0 10 

 Above solutions were prepared in Milli-Q Water 

2.8.5 Instrumental Parameters 

The above prepared standard solution, sample solutions and Milli-Q water were injected onto 

High Performance Liquid Chromatograph (HPLC) using validated parameters(JRF Study 

Number 228-2-13-5312). 

Instrument : HPLC 

Column : C-18 [250 mm x 4.6 mm (i.d.) x 5 μm particle size] 

Wave length : 260 nm 

Flow rate : 1.0 mL/minute 

Injection volume : 20 μL 

Mobile Phase : Acetonitrile (70): 0.1 % formic acid in water (30) v/v 

2.9 Procedure for LC-Mass Spectral Analysis 

2.9.1 Preparation of Sodium 3-nitrobenzenes sulfonate Reference Standard and Test Item 

Solutions 

Sample Weight (mg) Final Volume 

(mL) 

Identification Volume made 

using 

Reference Standard 25.47 25 A 

Milli Q Water 

Test Item 24.20 25 R1

The preparation of reference standard solution A (Refer Section 2.8.1 for Reference 

Standard) and R1 (Refer Section 2.8.4 for Test Item) 
JRF Study Number: 240-2-11-5551 (Final Report) Page 16 of 52 

2.9.2 LC Mass Parameters 

 The Above prepared solutions were analyzed on LC-MS using following parameters: 

LC-MS Parameters 

Instrument : LC-MSD SL 

Column : C-18, [150 mm x 4.6 mm (i.d.), 3.5 μm particle size] 

Wave Length : 260 nm 

Mobile Phase : Acetonitrile (70): 0.1 % Formic Acid (30) 

Flow Rate : 0.7 mL/minute 

Injection Volume : 20.0 μL 

MASS PARAMETERS 

Mass Range : 50 to 700 m/z 

Dry Gas Flow Rate : 10.0 L/minute 

Dry Gas Temperature : 350 °C 

Nebulizer Pressure : 50 psi 

Fragmentor Voltage : 100 - 125 Volts (negative) 

Quadra pole Temperature : 100 °C 

Capillary Voltage : 4000 Volts (positive and negative)

2.10 Procedure for NMR Analysis 

2.10.1 Preparation of Sodium 3-nitrobenzenes sulfonate Reference Standard and Test Item 

Solution 

 A quantity of 5 mg (approx.) sodium 3-nitrobenzene sulfonate reference standard and test item 

were transferred separately into the NMR probe tube and mixed with 1.0 mL deutorated methanol 

for NMR analysis using following parameter. 

NMR Parameters 

Instrument : Avance make Bruker (300 MHz) 

Atom : H1

 Range : Delta 0 – 10 

 Solvent : Deuterated Methanol (MeOD) 

Test Site 

NMR Spectrometer equipment at the Evotec India Limited, Thane, Mumbai, India. The test site 

selected for this study is not a member of National GLP Compliance Monitoring Program. The 

test site was selected in consultation with the study sponsor. Jai Research Foundation does not 

claim the GLP compliance for the test site and the part of study conducted.
JRF Study Number: 240-2-11-5551 (Final Report) Page 17 of 52 

3. RESULTS 

3.1 Infrared Spectra 

The IR spectral analysis sodium 3-nitrobenzene sulfonate reference standard and test item were 

confirmed their structure based on the presence of specific absorption bands due to the 

characteristic functional groups: 

Functional groups Reference Standard Sample 

- CH stretching 3106.76, 2936.09 and 2873.42 3102.9, 2935.13 and 2873.42 

- CH bending 1467.56 1468.53 

-C=C stretching in benzene ring 1611.23 1610.27 

-NO2 stretching 1536.99 and 1351.86 1536.02 and 1351.86 

-SO3 H stretching 1190.83, 1051.98 and 647.965 1192.76, 1047.16 and 670.142 

-adjacent H- atom 764.637 764.637 

3.2 Mass Analysis 

Characterization of sodium 3-nitrobenzene sulfonate was studied using LC-MS by injecting 

solutions of sodium 3-nitrobenzene sulfonate reference standard and test item. The 

characterization data based on molecular mass and retention time are summarized below: 

Sample Retention Time (min) Mass (m/z) 

Reference Standard 3.999 202.2 

Test Item 4.018 202.2 

3.3 NMR Analysis 

Identification of sodium 3-nitrobenzene sulfonate in reference standard and test item using NMR 

based on chemical shift and numbers of proton present in standard and test item are summarized 

below: 

Molecular Structure: 

SO3

NO2

H

H

H

H

Na

- +

1

2

3 4

Identification No of 

Protons Multiplicity Chemical Shift (δ), ppm Integration 

Standard Sample Standard Sample 

1 1 Triplet 7.724 – 7.764 7.723 – 7.764 1.09 1.09 

2 1 Doublet 8.216 –8.242 8.216 –8.242 1.06 1.06 

3 1 Doublet 8.327- 8.369 8.327- 8.369 1.11 1.18 

4 1 Singlet 8.666 – 8.675 8.665 – 8.675 1.00 1.00 

3.4 Active Ingredient Content 

Active Ingredient Content of sodium 3-nitrobenzene sulfonate was carried out using validate 

analytical method [Reference JRF Research Number: 228-2-13-5312]. 

The mean active Ingredient Content of sodium 3-nitrobenzene sulfonate was 96.88% w/w. 
JRF Study Number: 240-2-11-5551 (Final Report) Page 18 of 52 

4. CONCLUSION 

From the results of the presence of characteristic absorption bands revealed in IR spectra, 

molecular mass and the retention time obtained by LC-MS and the numbers of protons present 

revealed by NMR spectra the structure of sodium 3-nitrobenzene sulfonate was confirmed and it 

was concluded that the sodium 3-nitrobenzene sulfonate technical is similar to that of sodium 3-

nitrobenzene sulfonate reference standard. 
JRF Study Number: 240-2-11-5551 (Final Report) Page 19 of 52 

5. REFERENCES 

European Commission: Directorate General Health and Consumer Protection 2000: SANCO/ 

3030/99 rev. 4 “Technical and Material Preparations: Guidance for generating and reporting 

methods of analysis in support of pre-and post-registration data requirements for Annex II (Part A 

Section 4) and Annex III (part A, Section 5) of Directive 91/414/EEC” as amended by European 

Commission Directive 94/37/EC and 96/46/EC. 

JRF/PC/SOP-4090: Standard Operating Procedure for Spectra Analysis (IR, UV, Mass and 

NMR) of Test Item. 

Shrimali Asfak, 2012: “Validation of Analytical Method for Determination of Sodium 3-

Nitrobenzenesulfonate Active Ingredient Concentration In Dose Formulation”, JRF Research 

Number 228-2-13-5312, Jai Research Foundation, Valvada - 396 108, Gujarat,India. 

OECD, 1998: OECD Series on Principles of Good Laboratory Practice and Compliance 

Monitoring, Number 1, "OECD Principles on Good Laboratory Practice" ENV/MC/CHEM(98)17 

(as revised in 1997). 

Robert M. Silverstein, Francis X. Webster, 1997: Spectrometric Identification of Organic 

Compounds, Wiley, 6th edition. 

U.S. EPA, 1996: OPPTS 830.1800 “Enforcement of Analytical Method” (EPA 712-C-96-013). 

United States Environmental Protection Agency, Product Properties Test Guidelines (August 

1996). 

Williams Dudley H.; and Fleming Ian, Spectroscopic Methods in Organic Chemistry: Tata 

McGraw-Hill Publishing Company Limited, 4th Edition (1988). 
JRF Study Number: 240-2-11-5551 (Final Report) Page 20 of 52 

Characterization of Sodium 3-Nitrobenzene Sulfonate 

(CAS # 127-68-4, US EPA HPV 3) 

TABLE 1 

Linearity Data of Sodium 3-Nitrobenzene Sulfonate Reference Standard 

Concentration 

(mg/L) Replications Peak Area Counts Mean Peak Area Counts % Variation

24.96 

R1 977603 

979493.00 0.39 

R2 981383 

49.92 

R1 1964680 

1965925.50 0.13 

R2 1967171 

74.88 

R1 3036694 

3033790.00 0.19 

R2 3030886 

99.84 

R1 3994671 

3991843.50 0.14 

R2 3989016 

124.80 

R1 4969853 

4971502.50 0.07 

R2 4973152 

149.76 

R1 5920712 

5921661.50 0.03 

R2 5922611 

Typical Calculation 

 Maximum Peak Area − Minimum Peak Area 

% Variation = --------------------------------------------------------- × 100 

 Maximum Peak Area 

 981383-977603 

= ----------------------- × 100 

 981383 

= 0.39% 

FIGURE 1: Linearity Curve of Sodium 3-Nitrobenzene Sulfonate Reference Standard 

0 25 50 75 100 125 150

Concentration (mg/L)

0

900

1800

2700

3600

4500

5400

Mean Peak Area (Thousands)

Intercept with y-axis (a) = 8806.63

Slope of the line (b) = 39704.24

Correlation co-efficient or 'r' value = 0.999

Equation : Y = bX + a

Y = 39704.24X + 8806.63
JRF Study Number: 240-2-11-5551 (Final Report) Page 21 of 52 

Characterization of Sodium 3-Nitrobenzene Sulfonate 

(CAS # 127-68-4, US EPA HPV 3) 

TABLE 2 

Calculation of Active Ingredient Content 

Replication 

Weight 

(mg) of 

Sample 

(W) 

N° of 

Injection 

Peak Area of 

Sample 

(R) 

Weight 

(mg) of 

Standard

(W1) 

Peak Area of 

Standard 

Mean Peak Area 

of Standard 

(R1) 

A.I. 

Content 

(% w/w)

Mean 

A.I. 

Content

(% w/w)

R1 24.20 

I 3747663 

25.47 

3993545 

3994632.75 

96.77 

96.91 

II 3758360 3997020 97.04 

R2 24.34 

I 3762143 3990825 96.58 

96.85 

II 3783260 3997141 97.12 

Mean 96.88 

Purity of Standard (P) 98.00% Dilution Factor (D) 1.00 

Typical Calculation 

Sodium 3-Nitrobenzene Sulfonate A.I. Content (% w/w) 

 R × W1 × P 

 = ------------------- × D 

 R1 × W 

 3747663 × 25.47 × 98.00 

 = --------------------------------- × 1.00 

 3994632.75 × 24.20 

= 96.77% w/w 
JRF Study Number: 240-2-11-5551 (Final Report) Page 22 of 52 

Characterization of Sodium 3-Nitrobenzene Sulfonate 

(CAS # 127-68-4, US EPA HPV 3) 

APPENDIX 1 

IR, Mass, NMR Spectra and HPLC Chromatograms 
JRF Study Number: 240-2-11-5551 (Final Report) Page 23 of 52 

APPENDIX 1 (Continued) 

 
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APPENDIX 1 (Continued) 

 
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APPENDIX 1 (Continued) 
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APPENDIX 1 (Continued) 
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APPENDIX 1 (Continued) 
JRF Study Number: 240-2-11-5551 (Final Report) Page 28 of 52 

APPENDIX 1 (Continued) 
JRF Study Number: 240-2-11-5551 (Final Report) Page 29 of 52 

APPENDIX 1 (Continued) 
JRF Study Number: 240-2-11-5551 (Final Report) Page 30 of 52 

APPENDIX 1 (Continued) 
JRF Study Number: 240-2-11-5551 (Final Report) Page 31 of 52 

APPENDIX 1 (Continued) 

 
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APPENDIX 1 (Continued) 
JRF Study Number: 240-2-11-5551 (Final Report) Page 33 of 52 

APPENDIX 1 (Continued) 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 34 of 52 

APPENDIX 1 (Continued) 
JRF Study Number: 240-2-11-5551 (Final Report) Page 35 of 52 

Characterization of Sodium 3-Nitrobenzene Sulfonate 

(CAS # 127-68-4, US EPA HPV 3) 

APPENDIX 2 

Signed Study Plan and Study Plan Amendment 

JRF Study Number: 240-2-11-5551 Page 1 of 13 

STUDY PLAN 

CHARACTERIZATION OF SODIUM 3-NITROBENZENE SULFONATE

(CAS # 127-68-4, US EPA HPV 3) 

GUIDELINES: SANCO/ 3030/99 rev. 4 AND U.S. EPA OPPTS 830.1800 

TEST RULE: 40 CFR 799.5089 

SPONSOR 

TIARCO CHEMICAL 

1300 TIARCO DRIVE 

DALTON 

 GA 30720 

TECHNICAL CONTACT: JEFF VANDERLIP 

REGULATORY CONTACT: BENNETT KING 

STUDY DIRECTOR: NIRAL M. BHANDARI 

TEST FACILITY 

JAI RESEARCH FOUNDATION 

DEPARTMENT OF CHEMISTRY 

VALVADA - 396 108 

DIST. VALSAD 

GUJARAT 

INDIA 

OCTOBER - 2012 
JRF Study Number: 240-2-11-5551 (Final Report) Page 36 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 2 of 13 

CONTENTS 

PAGE N° 

TITLE PAGE ........................................................................................................................................................... 1

CONTENTS............................................................................................................................................................. 2 

1. GENERAL INFORMATION ........................................................................................................................... 3 

 1.1 Study Professional ................................................................................................................................. 3 

 1.2 Test Facility Management ...................................................................................................................... 3 

 1.3 Technical Contact .................................................................................................................................. 3 

 1.4 Regulatory Contact ................................................................................................................................ 3 

 1.5 Study Schedule ....................................................................................................................................... 4 

 1.6 Study Plan and Amendment (if any) Distribution .................................................................................. 4 

2. INTRODUCTION ............................................................................................................................................ 4 

 2.1 Objective ................................................................................................................................................ 4 

 2.2 Regulatory Guidelines ............................................................................................................................ 4 

 2.3 Test Item ................................................................................................................................................ 5 

 2.4 Reference Standard ................................................................................................................................ 6 

 2.5 Reference Standard Retention ................................................................................................................ 6 

3. GOOD LABORATORY PRACTICE (GLP) ................................................................................................... 6 

 3.1 GLP Compliance .................................................................................................................................... 6 

 3.2 Standard Operating Procedures (SOPs) ................................................................................................. 7 

 3.3 Amendment to Study Plan ..................................................................................................................... 7 

 3.4 Deviation from the Study Plan ............................................................................................................... 7 

 3.5 Quality Assurance .................................................................................................................................. 7 

4. EXPERIMENTAL PROCEDURE ................................................................................................................... 7 

 4.1 Equipment/Instruments .......................................................................................................................... 7 

 4.2 Solvents and Chemicals ......................................................................................................................... 7 

 4.3 Sample Preparations for IR Test ............................................................................................................ 7 

 4.3.1 Interpretation of IR Spectra ..................................................................................................... 8 

 4.4 Methodology for Mass Analysis [by LC-MS] ....................................................................................... 8 

 4.4.1 Preparation of Reference Standard / Test Item Solution for Mass Spectra .............................. 8 

 4.4.2 Mass Parameters ...................................................................................................................... 8 

 4.4.3 Mass Interpretation and Reporting ........................................................................................... 9 

 4.5 Methodology for NMR Analysis ........................................................................................................... 9 

 4.6 Active Ingredient Content ...................................................................................................................... 9 

 4.6.1 Preparation of Reference Standard Stock Solution .................................................................. 9 

 4.6.2 Linearity ................................................................................................................................... 9 

 4.6.3 Preparation of Sample Solution ............................................................................................. 10 

 4.6.4 Instrumental Parameters ........................................................................................................ 10 

 4.6.5 Calculation of Active Ingredient Content (% w/w) ............................................................... 10 

5. REPORT ......................................................................................................................................................... 11 

6. ARCHIVES .................................................................................................................................................... 11 

7. REFERENCES ............................................................................................................................................... 11 

8. STUDY PLAN APPROVAL .......................................................................................................................... 13
JRF Study Number: 240-2-11-5551 (Final Report) Page 37 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 3 of 13 

1. GENERAL INFORMATION

1.1 Study Professional 

 Note: List of additional study personnel will be documented in the study file and will be presented in the final report.

1.2 Test Facility Management : Hemant Desai 

 Assistant Director, Department of Chemistry 

 Jai Research Foundation, Off. N.H. N° 8 

 Near Daman Ganga Bridge 

 Valvada. 396 108, Dist. Valsad, Gujarat, India. 

 Ph.: + 91-260 654 0242 (Office) 

 Ph.: +91 99099 00940 (Mobile) 

 E-mail: desaihs@jrfonline.com 

1.3 Technical Contact : Jeff Vanderlip

 706/277-1300 

 Tiarco Chemical 

 1300 Tiarco Drive 

 Dalton. Georgia 30720 

1.4 Regulatory Contact : Bennett King 

 864/457-2321 

 Tiarco Chemical 

 1300 Tiarco Drive 

 Dalton. Georgia 30720 

 

Name of Personnel Qualification Profession Experience Training 

Niral M. Bhandari 

(Study Director) 

M.Sc. Chemist 6 years Declared Study Director for all 

Physico-chemical studies 

A.H. Patel 

(Dy. Study Director) 

M.Sc. Chemist 14 years Declared Study Director for all 

Physico-chemical studies 
JRF Study Number: 240-2-11-5551 (Final Report) Page 38 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 4 of 13 

1.5 Study Schedule 

 Study Initiation Date : October 12, 2012 

 Experiment Start Date : October 15, 2012 

 Experiment Completion : Latest by October, 2012 

 Draft Report Submission : Latest by October, 2012 

Study Completion : Within two weeks from the date of receipt of the comments on 

the final draft report from the Sponsor. 

1.6 Study Plan and Amendment (if any) Distribution 

 a. Original copy in Archive and Study Sponsor; b. Photocopy to Study Director and QAU 

2. INTRODUCTION 

2.1 Objective 

The objective of this study is to determine IR, Mass, NMR and active ingredient content of sodium 3-

nitrobenzene sulfonate (CAS N° 127-68-4). 

2.2 Regulatory Guidelines 

European Commission: Directorate General Health and Consumer Protection 2000: SANCO/ 3030/99 rev. 

4 “Technical and Material Preparations: Guidance for generating and reporting methods of analysis in 

support of pre- and post-registration data requirements for Annex II (Part A Section 4) and Annex III (part 

A, Section 5) of Directive 91/414/EEC” as amended by European Commission Directive 94/37/EC and 

96/46/EC. 

U.S. EPA, 1996: OPPTS 830.1800 “Enforcement of Analytical Method” (EPA 712-C-96-013). United 

States Environmental Protection Agency, Product Properties Test Guidelines (August 1996). 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 39 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 5 of 13 

2.3 Test Item 

The Test Item Data Sheet has been completed by the Sponsor. The representative sample of the test item 

will be retained for archive purpose. Any residual test item will be disposed of at JRF after the expiry date 

unless otherwise instructed by the Sponsor. The details relating to the test item (as furnished by the study 

Sponsor) are as follows: 

Test Item Name 

IUPAC Name 

C.A. Name 

CAS N° 

Batch N° 

Analysed Purity 

Manufacture by 

Supplied by 

Date of Manufacture 

Date of Expiry 

Appearance 

Sodium 3-nitrobenzene sulfonate 

Benzenesulfonic acid, 3-nitro, sodium salt (1:1) 

Benzenesulfonic acid, 3-nitro, sodium salt (1:1) 

127-68-4 

50623 

96.9% (Information provided by sponsor via COA) 

Tiarco Chemical 

Tiarco Chemical 

September 08, 2011 

September 08, 2013 

Yellow to tan granular 

Data related to the characterization of the test item are documented in a Certificate of analysis provided 

by the Sponsor and will be found in annexes of the study report. The test item information and analytical 

identification provided by the Sponsor will be responsibility of the Sponsor.

Storage Condition 

(at JRF)

As per the instruction received from the Sponsor on storage of the test item, 

the test item will be stored in its original container, as supplied by the Sponsor 

at 40 - 90 °F in Test Item Control Office (TICO). The stability of the test item 

in storage will be the responsibility of the Sponsor. 

JRF Test Item Code SNB 444 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 40 of 52 

APPENDIX 2 (Continued)

O2N SO3H

.Na

JRF Study Number: 240-2-11-5551 Page 6 of 13 

2.4 Reference Standard 

Details of the reference standard are as follows:

Reference Standard 

IUPAC Name 

C.A. Name 

Molecular Weight 

Molecular Formula 

CAS N° 

Lot N° 

Date of Receipt 

Date of Expiry 

Purity 

Source 

JRF Entry Number 

Storage Condition 

Sodium 3-Nitrobenzenes sulfonate 

Benzenesulfonic acid, 3-nitro-, sodium salt (1:1) 

Benzenesulfonic acid, 3-nitro-, sodium salt (1:1)

225.15 

O2NC6H4SO3Na 

[127-68-4] 

07315KNV 

August 07, 2012 

August 06, 2017 (as per JRF SOP-152) 

98.0% 

Sigma-Aldrich, USA 

CPC-54 

Room Temperature 

 Source: Molecular structure was obtained from Internet. 

2.5 Reference Standard Retention 

 The reference standard will be retained at the Department of Chemistry till the declared expiry or 

 re assay date.

3. GOOD LABORATORY PRACTICE (GLP) 

3.1 GLP Compliance 

This study will be conducted in compliance with the OECD Principles of Good Laboratory Practice (as 

revised in 1997), ENV/MC/CHEM(98)17, N° 1, Environment Directorate, the Organisation for Economic 

Co-operation and Development, Paris (1998) and all subsequent OECD consensus documents. 

The NMR spectra analysis will be performed at the Evotec India Limited, Thane, Mumbai, India (Test 

Site). The test site selected for this study is not a member of National GLP Compliance Monitoring 

Program. The test site is selected in consultation with the Study Sponsor. Jai Research Foundation does not 

claim the GLP compliance for the test site and the part of study to be conducted. 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 41 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 7 of 13 

3.2 Standard Operating Procedures (SOPs) 

Unless otherwise specified all procedures mentioned in the study plan are subject to detailed Standard 

Operating Procedures of Jai Research Foundation. 

3.3 Amendment to Study Plan 

This study plan may be subject to amendment. Amendment to study plan, whether initiated by the Sponsor 

or the Study Director will be generated, authorized by the Study Director and will be sent to the Sponsor 

for approval. 

In the event that circumstances dictate immediate action, the nature of these circumstances will 

communicated to the Sponsor as soon as practicable (by telephone, facsimile transmission or e-mail) and 

will be confirmed as soon as possible by way of formal study plan amendment. 

3.4 Deviation from the Study Plan 

Any deviation from the study plan will be documented in study file and reported in the study report. 

3.5 Quality Assurance 

This study plan has been verified by JRF QAU and documented (Number 48657). The Quality Assurance 

Unit (QAU) of JRF will audit/inspect the critical phase(s) of the study, the raw data, draft and final reports. 

The audit/inspection reports will be provided to the Study Director and the Test Facility Management. The 

date of audits/inspections and reporting of findings to the Study Director and the Test Facility Management 

will be incorporated in the study report.

4. EXPERIMENTAL PROCEDURE 

4.1 Equipment/Instruments 

 HPLC, FT-IR Spectrometer, LC-MS, NMR and Balance. 

4.2 Solvents and Chemicals 

 Acetonitrile, methanol, Milli-Q water and potassium bromide. 

4.3 Sample Preparations for IR Test 

A known quantity of 2.0 mg reference standard and test item will be mixed with 100 mg (approx.) dried 

potassium bromide. The mixture will be grinded finely using pestle and mortar and the mixture was placed 

in DRA sample holder of sample compartment. The IR spectrum will be recorded. The background 

spectrum will be recorded before analyzing the sample by using blank KBr. 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 42 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 8 of 13 

 All samples and background spectra will be recorded using the following parameters: 

 Instrument : FT-IR Spectrometer 

 Wave Number Range : 400-4000 cm-1 

 Resolution : 4.0 cm-1 

4.3.1 Interpretation of IR Spectra 

The spectral data interpretation will be performed using the standard references viz., Williams Dudley H. 

(1988) and Robert M. Silverstein, Francis X. Webster, (1997).

4.4 Methodology of Mass Analysis [by LC-MS] 

4.4.1 Preparation of Reference Standard/Test Item Solution for Mass Spectra 

The known quantities of sodium 3-nitrobenzene sulfonate reference standard and test item will be weighed 

in into separate volumetric flasks of 10 mL capacity, contents will be dissolved in 5.0 mL Milli-Q water 

and the volume will be made upto the mark with Milli-Q water. The sample solutions will be injected onto 

LC-MS in duplicate. 

4.4.2 Mass Parameters 

 The Above prepared solution were analysed on LC-MSD SL using following parameters: 

 LC-MS Parameters 

 Instrument : LC-MSD SL 

 Column : C-18, [150 mm x 4.6 mm (i.d.), 3.5 μm particle size] 

 Wave Length : 260 nm 

 Mobile Phase : Acetonitrile (70): 0.1 % formic acid in water (30) v/v 

 Flow Rate : 0.7 mL/minute 

 Injection Volume : 10.0 μL 

 MASS PARAMETERS 

 Mass Range : 50 to 700 m/z 

 Dry Gas Flow Rate : 10.0 L/minute 

 Dry Gas Temperature : 350 °C 

 Nebulizer Pressure : 50 psi 

 Fragmentor Voltage : 100 - 125 Volts (negative) 

 Quadra pole Temperature : 100 °C 

 Capillary Voltage : 4000 Volts (positive and negative) 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 43 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 9 of 13 

4.4.3 Mass Interpretation and Reporting 

The mass interpretation will be based on comparison of retention time, elution pattern and 

mass/fragmentation pattern of standard and test item and the retention time and molecular mass will be 

reported 

4.5 Methodology for NMR Analysis 

A quantity of 5 mg (approx.) reference standard and test item will be weighed and transferred into the 

NMR probe tube and mixed with approx. 1.0 mL in suitable solvent for NMR analysis using following 

parameters:

 NMR Parameters 

 Instrument : Avance make Bruker (300 MHz) 

 Atom : H1

 Range : Delta 0 – 10 

 Solvent : Deuterated Oxide (D2O) 

 Test Site 

NMR Spectrometer equipment at the Evotec India Limited, Thane, Mumbai, India. The test site selected for 

this study is not a member of National GLP Compliance Monitoring Program. The test site is selected in 

consultation with the Study Sponsor. Jai Research Foundation does not claim the GLP compliance for the 

test site and the part of study to be conducted. 

4.6 Active Ingredient Content 

4.6.1 Preparation of Reference Standard Stock Solution 

A known quantity of sodium 3-nitrobenzene sulfonate reference standard of known purity will be weighed 

into a volumetric flask of known capacity. The contents will be dissolved in a small volume of Milli-Q 

water and the volume will be made upto the mark with Milli-Q water. The various concentrations of 

reference standard solution will be prepared by dilution of stock solution in Milli-Q water for linearity. 

4.6.2 Linearity 

Linearity of the method will be carried out before analysing the samples. Six different concentrations of 

sodium 3-nitrobenzene sulfonate reference standard will be injected onto HPLC in duplicate. The linear 

calibration curve will be established by plotting the mean peak areas against concentrations (mg/L). The 

correlation coefficient (r), intercept (a) and slope (b) will be calculated. 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 44 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 10 of 13 

4.6.3 Preparation of Sample Solution 

A known quantity of test item will be weighed into a volumetric flask of known capacity. The contents will 

be dissolved in a small volume of Milli-Q water and the volume will be made upto the mark with Milli-Q 

water. The solutions will be filtered (if required) and diluted (if required) using Milli-Q water. 

4.6.4 Instrumental Parameters 

The reference standard solutions, sample solutions and Milli-Q water (solvent used for standard and sample 

solution preparation) will be injected onto a High Performance Liquid Chromatograph (HPLC) using 

validated instrument parameters instrumental parameters [Reference JRF Research Number: RES-2-13-

5312]. 

Instrument : HPLC 

Column : C-18 [250 mm x 4.6 mm (i.d.) x 5 μm particle size] 

Wave length : 260 nm 

Flow rate : 1.0 mL/minute 

Injection volume : 20 μL 

Mobile Phase : Acetonitrile (70): 0.1 % formic acid in water (30) v/v 

4.6.5 Calculation of Active Ingredient Content (% w/w) 

The sodium 3-nitrobenzene sulfonate content will be calculated using the following formula: 

 R × W1 × P 

= -------------- × D 

 R1 × W 

Where, 

 R = Peak area of the sample 

 R1 = Mean peak area of the reference standard 

 W = Weight (mg) of the sample 

 W1 = Weight (mg) of the reference standard 

 P = Purity (%) of reference standard 

 D = Dilution factor 

Note: Active Ingredient content will be conducted in triplicate.

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 45 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 11 of 13 

5. REPORT 

Unless otherwise requested by the Sponsor, one copy of the final report will be issued along with one soft 

copy in PDF. The report will include the following information: 

- Description of the test item and reference standard including certificates of analysis 

- Complete details of the experimental procedure 

- IR spectra with peak tables 

- Mass Spectra. 

- NMR Spectra 

- Spectral interpretation 

- Summary of observations 

- Results 

- Chromatograms 

- Personnel involved in the study 

- The signed study plan and study plan amendment(s) (if any) 

- Record of deviation(s) from the study plan (if any) 

6. ARCHIVES 

On completion of the study all original raw data including any storage medium for electronically 

recorded data, spectra, documentation, the signed study plan, study plan amendment (if any), the draft 

report, a copy of final report and the representative sample of the test item will be retained in the GLP 

Archives at Jai Research Foundation for a period of ten years. At the end of this period, the Sponsor's 

instructions will be sought to either extend the archiving period or return the archived material to the 

Sponsor or for the material to be disposed of. 

7. REFERENCES 

European Commission: Directorate General Health and Consumer Protection 2000: SANCO/ 3030/99 

rev. 4 “Technical and Material Preparations: Guidance for generating and reporting methods of analysis 

in support of pre-and post-registration data requirements for Annex II (Part A Section 4) and Annex III 

(part A, Section 5) of Directive 91/414/EEC” as amended by European Commission Directive 94/37/EC 

and 96/46/EC. 

Shrimali A., 2012: “Validation of Analytical Method for Determination of Sodium 3-

Nitrobenzenesulfonate Active Ingredient Concentration In Dose Formulation”, JRF Research Number 

228-2-13-5312, Jai Research Foundation, Valvada - 396 108, Gujarat, India. 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 46 of 52 

APPENDIX 2 (Continued)

JRF Study Number: 240-2-11-5551 Page 12 of 13 

OECD, 1998: OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, 

Number 1, "OECD Principles on Good Laboratory Practice" ENV/MC/CHEM(98)17 (as revised in 

1997). 

Robert M. Silverstein, Francis X. Webster, 1997: Spectrometric Identification of Organic Compounds, 

Wiley, 6th edition. 

U.S. EPA, 1996: OPPTS 830.1800 “Enforcement of Analytical Method” (EPA 712-C-96-013). United 

States Environmental Protection Agency, Product Properties Test Guidelines (August 1996). 

Williams Dudley H.; and Fleming Ian, Spectroscopic Methods in Organic Chemistry: Tata McGraw-Hill 

Publishing Company Limited, 4th Edition (1988). 

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 47 of 52 

APPENDIX 2 (Continued)

 
JRF Study Number: 240-2-11-5551 (Final Report) Page 48 of 52 

APPENDIX 2 (Continued)
JRF Study Numbe

Certific

r: 240-2-11-

Characte

cate of Analy

-5551 (Final R

erization of 

(CAS # 12

ysis of Sodium

Report)

Sodium 3-N

7-68-4, US E

APPENDIX

m 3-Nitroben

Nitrobenzen

EPA HPV 3

X 3 

nzenes Sulfon

ne Sulfonate

3) 

nate Technic

Page

e 

al Sample 

e 49 of 51 
JRF Study Number: 240-2-11-5551 (Final Report) Page 50 of 52 

Characterization of Sodium 3-Nitrobenzene Sulfonate 

(CAS # 127-68-4, US EPA HPV 3) 

APPENDIX 4 

Certificate of Analysis of Sodium 3-Nitrobenzenes Sulfonate Standard 
JRF Study Number: 240-2-11-

Characte

5551 (Final R

rization of 

(CAS # 12

GLP End

Report)

Sodium 3-N

7-68-4, US E

APPENDIX

orsement of 

Nitrobenzen

EPA HPV 3

X 5 

Compliance

ne Sulfonate

3) 

Page

 

 51 of 52 
JRF Study Number: 240-2-11-5551 (Final R

APPE

eport)

NDIX 5 (Continued) 

Page 52 of 52