JRF Study Number: 228-2-13-5312 (Final Report) Page 2 of 43 

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 Tiacro Drive

Dalton, GA 30720

Signature __________________________________

Date __________________________________



JRF Study Number: 228-2-13-5312 (Final Report) Page 6 of 43 

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 ............................................................................................. 9 

SUMMARY ................................................................................................................................................ 10 

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

 1.1 Study Objective .......................................................................................................................... 11 

 1.2 Study Guidelines ........................................................................................................................ 11 

 1.3 Test Facility and Study Period ................................................................................................... 11 

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

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

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

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

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

2.4 Solvents and Chemicals .............................................................................................................. 13 

 2.5 Principle...................................................................................................................................... 13 

2.6 Validation of Analytical Method for Determination of Sodium 3-Nitrobenzensulfonate 

 using HPLC ............................................................................................................................... 13 

 2.6.1 Specificity .......................................................................................................................... 13 

 2.6.1.1 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard 

 Stock Solution ............................................................................................... 13 

 2.6.1.2 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard 

 Working Solutions ........................................................................................ 13 

2.6.1.3 Preparation of Test Item Stock Solution ....................................................... 14 

2.6.1.4 Preparation of Test Item Working Solution in Dose Formulation ................ 14 

 2.6.2 Instrumental Parameters ................................................................................................ 14 

 2.6.3 Limit of Detection (LOD) ............................................................................................ 14 

 2.6.3.1 Preparation of Test Item Solutions in Dose Formulation ............................... 14 
JRF Study Number: 228-2-13-5312 (Final Report) Page 7 of 43 

CONTENTS (Continued) 

 PAGE N°

2.6.4 Linear Dynamic Range ................................................................................................. 15 

 2.6.4.1 Preparation of Sodium 3-Nitrobenzensulfonate Working Standard Solutions 15 

 2.6.5 Precision (% RSD) and Accuracy (% Recovery) ......................................................... 15 

 2.6.5.1 Determination of Homogeneity of Mixing and Stability Sample in Dose 

 Formulation ................................................................................................... 15 

 2.6.6 Calculation .................................................................................................................... 16

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

3.1 Specificity................................................................................................................................... 17

3.2 Linear Dynamic Range .............................................................................................................. 17

3.3 Limit of Quantitation ................................................................................................................. 17

3.4 Precision (% RSD) .................................................................................................................... 17

3.5 Accuracy (% Recovery) ............................................................................................................ 18

3.6 Homogeneity and Stability ........................................................................................................ 18

4. CONCLUSION.................................................................................................................................... 19

5. REFERENCES..................................................................................................................................... 20

TABLES

1. Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate for Precision (%RSD) and 

 Accuracy (% Recovery) and the Level of Test Concentration during 

 Toxicology Study (0 hr) ............................................................................................................ 21 

2. Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate at the Level of 

 Test Concentration during Toxicology Study (24 h) ................................................................. 22 

3. Limit of Quantitation of Sodium 3-Nitrobenzensulfonate in Dose Formulation ....................... 23

4. Calculation of Precision (% RSD) and Accuracy (% Recovery) of the Level of Test Concentration 

during Toxicology Study for Sodium 3-Nitrobenzensulfonate in Dose Formulation (0 hr)............. 24

5. Calculation of (%) Recovery and Stability at the Level of Test Concentration during 

Toxicology Study for Sodium 3-Nitrobenzensulfonate in Dose Formulation (24h).................. 25
JRF Study Number: 228-2-13-5312 (Final Report) Page 8 of 43 

CONTENTS (Continued) 

PAGE N°

FIGURES 

 1. Linear Dynamic Curve for Sodium 3-Nitrobenzensulfonate for Precision (%RSD) and Accuracy 

 (% Recovery) and the Level of Test Concentration during Toxicology Study (0 hr) ............... 21 

2. Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate at the Level of Test Concentration 

 during Toxicology Study (24 h) ................................................................................................ 22 

APPENDICES 

1. Chromatograms .......................................................................................................................... 26

2. Signed Study Plan and Study Plan Amendment ........................................................................ 29

 3. Certificate of Analysis of Test Item .......................................................................................... 40

 4. Certificate of Analysis of Reference Standard of Sodium 3-Nitrobenzensulfonate .................. 41 

 5. GLP Endorsement of Compliance .............................................................................................. 42

JRF Study Number: 228-2-13-5312 (Final Report) Page 10 of 43 

SUMMARY 

This study was conducted to validate the high performance liquid chromatographic (HPLC) analytical 

method for the determination of active ingredient concentration of Sodium 3-Nitrobenzensulfonate in dose 

formulation. The test item was supplied by Tiarco Chemical, 1300 Tiacro Drive, Dalton, GA 30720. The

method followed was as per the guidelines of U.S. EPA OPPTS 860.1340 (August, 1996) and 

SANCO/3029/99 rev.4.

The analytical method for the determination of active ingredient concentration of Sodium 3-

Nitrobenzensulfonate was validated by analysing the test item and reference standard. The validation covered 

the aspects namely: (i) specificity, (ii) linear dynamic range (LDR), (iii) limit of quantitation (LOQ), (iv) 

precision (% RSD) and accuracy (% Recovery).

The method validation are summarised below:

Parameters Results

Dose Formulation Dose Formulation (RO Water)

Active ingredient Sodium 3-Nitrobenzensulfonate

Specificity (Non-analyte interference) No interference

Linear Dynamic Range 

(LDR)

Concentration Range (mg/L)

Intercept (a)

Slope of the line (b)

Correlation Coefficient (r)

10.04 to 100.35

12827.10

40335.43

0.999

Limit of Quantitation (LOQ) [mg/L] 0.5

Precision (% RSD)

Fortification Level -

5 mg/mL 0.07

% RSD [Horwitz equation] 2.99

100 mg/mL 0.85

% RSD [Horwitz equation] 1.90

Accuracy (Recovery %)

5 mg/mL 101.98

100 mg/mL 99.60

Homogeneity and 

Stability (Recovery %)

24 Hours

5 mg/mL 101.87

100 mg/mL 99.28
JRF Study Number: 228-2-13-5312 (Final Report) Page 11 of 43 

1. INTRODUCTION 

1.1 Study Objective

This study was performed to validate the HPLC analytical method for determination of Sodium 3-

Nitrobenzensulfonate active ingredient concentration in dose formulation. The study was 

conducted in compliance with OECD Principles of GLP (1998). 

1.2 Study Guidelines 

The present study was conducted according to the following guidelines: 

U.S. EPA, 1996: OPPTS 860.1340 “Residue Analytical Method”. United States Environmental 

Protection Agency, Residue Chemistry Test Guidelines, EPA 712-C-96-174 (August 1996), 

and 

European Commission 2000: Residues: Guidance for generating and reporting methods of analysis 

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

(part A, Section 5) of Directive 91/414; SANCO/3029/99 rev. 4 of 11 July 2000."Directorate 

General Health and Consumer Protection". 

1.3 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 : August 25, 2012 

Experiment Start : August 30, 2012 

Experiment Completion : August 31, 2012 

Study Completion : October 11, 2012 

1.4 Archives 

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

documentation, chromatograms, the signed study plan, 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: 228-2-13-5312 (Final Report) Page 12 of 43 

2. EXPERIMENTAL PROCEDURE 

2.1 Test Item 

Details of Test Item Provided by the Sponsor 

Test Item Name

IUPAC Name

CA Name

CAS Number

Batch/Lot N°

Concentration / Purity

Supplied by 

Manufactured by

Date of Manufacture

Date of Expiry

Appearance / Colour 

Sodium 3-Nitrobenzensulfonate 

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

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

127-68-4

50623

96.9 % (Certificate of Analysis, Refer Appendix - 3)

Reservol LP

Tiarco Chemical

August 09, 2011

August 09, 2013

Yellow to tan granular solid

Storage Condition (at JRF) As per the instruction received from the Sponsor on storage of 

the test item, the test item was stored in its original container as 

supplied by the Sponsor in room temperature (40 to 90 °F) 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 

 Details of the Reference Standard 

Reference Standard

C.A. Name 

Molecular Weight

Molecular Formula

CAS N°

LOT N°

Date of Receipt

Date of Expiry

Purity 

Source

Storage Condition 

JRF Entry Number

Sodium 3-Nitrobenzensulfonate 

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% (Certificate of Analysis, Refer Appendix - 4)

ALDRICH

Room Temperature

CPC-54
JRF Study Number: 228-2-13-5312 (Final Report) Page 13 of 43 

2.3 Instruments/Equipment 

Sr. N° Instrument/Apparatus Model Make / Supplier

1 Analytical Balance GR-202 Adair Dutt 

2 HPLC 2010 CHT Shimadzu

2.4 Solvents and Chemicals 

Sr. N° Name Grade Source

1 Acetonitrile HPLC s.d. fine-chem ltd.

2 Formic acid HPLC Meark

3 Water Milli-Q Milli-Q water system

2.5 Principle 

The active ingredient concentration of Sodium 3-Nitrobenzensulfonate is determined by high 

performance liquid chromatograph on a reversed phase column (C-18) kromosil using acetonitrile : 

0.1% formic acid in water (70: 30), v/v as mobile phase and UV detection at 260 nm. 

2.6 Validation of Analytical Method for Determination of Sodium 3-Nitrobenzensulfonate using 

 HPLC 

The analytical method for the determination of active ingredient concentration of Sodium 3-

Nitrobenzensulfonate in dose formulation was validated. The validation covered the aspects 

namely: (i) specificity, (ii) linear dynamic range (LDR), (iii) limit of quantitation (LOQ), (iv) 

precision (% RSD) and accuracy (% Recovery). 

2.6.1 Specificity 

2.6.1.1 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard Stock Solution 

 A quantity of 10.24 mg Sodium 3-Nitrobenzensulfonate (98.0% purity) was weighed in a volumetric 

flask of 100 mL capacity, dissolved in 10 mL RO water and the volume was made upto the mark with 

RO water. The concentration of Sodium 3-Nitrobenzensulfonate in the stock solution was 100.35 

mg/L (standard stock solution RS). 

2.6.1.2 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard Working Solutions 

A volume of 1.0 mL stock solution RS was transferred into volumetric flask of 10 mL capacity and 

the volume was made upto the mark with RO water. The solution concentration of Sodium 3-

Nitrobenzensulfonate was 10.04 mg/L [working solution (L6)]. 
JRF Study Number: 228-2-13-5312 (Final Report) Page 14 of 43 

2.6.1.3 Preparation of Test Item Stock Solution 

A quantity of 10.34 mg Sodium 3-Nitrobenzensulfonate technical (96.9% purity) was weighed in a 

volumetric flask of 100 mL capacity and the volume was made upto the mark with dose formulation 

(RO water). The concentration of Sodium 3-Nitrobenzensulfonate in the stock solution was 100.19 

mg/L (Test Item stock solution, TI). 

2.6.1.4 Preparation of Test Item Working Solution in Dose Formulation 

A volume of 1.0 mL test item stock solution TI was transferred into volumetric flask of 10 mL 

capacity and the volume was made upto the mark with RO water. The solution concentration of 

Sodium 3-Nitrobenzensulfonate was 10.02 mg/L [Test Item working solution (STI)].

The blank RO water, Sodium 3-Nitrobenzensulfonate reference standard working solution (L6) and

test item working solution (STI) were injected onto HPLC in accordance with section 2.6.2 

2.6.2 Instrumental Parameters [Reference JRF Research Number : RES-2-12-5315]

Instrument : HPLC 

Column : C-18 kromosil [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 

Retention Time : 4.0 min. (Appox.) 

2.6.3 Limit of Quantitation (LOQ) 

2.6.3.1 Preparation of Test Item Solutions in Dose Formulation 

Stock Solution

Solution 

Taken

(mL)

Fina Volume

(mL)

Volume 

made up 

with 

Concentration 

Obtained 

(μg/mL)

Identification 

of 

Standard

Solution

TI

(100.19 mg/L) 1.0 10

RO water

10.02 LOQTI

LOQTI

(10.02 mg/L)

0.1 10 0.10 LOQTI1

0.5 10 0.50 LOQTI2

1.0 10 1.00 LOQTI3

1.5 10 1.50 LOQTI4

The test item solutions (LOQTI1, LOQTI2, LOQTI3 and LOQTI4) were injected onto the HPLC in 

 duplicate. The minimum concentration which could be quantified with signal to noise ratio (S/N) 

 10:1 was considered as LOQ. The average signal: noise ratio was calculated the noise obtained in 

 RO water untreated control dose formulation. 
JRF Study Number: 228-2-13-5312 (Final Report) Page 15 of 43 

2.6.4 Linear Dynamic Range 

2.6.4.1 Preparation of Sodium 3-Nitrobenzensulfonate Working Standard Solutions 

Standard Solution Solution 

Taken (mL)

Volume make 

upto (mL)

Obtained

Concentration (mg/L)

Solution

Standard 

Solution

RS

(100.35 mg/L)

- - 100.35 L6

8.0 10.0 80.28 L5

6.0 10.0 60.21 L4

4.0 10.0 40.14 L3

2.0 10.0 20.07 L2

1.0 10.0 10.04 L1

Above standard solutions were diluted with RO water of untreated control dose formulation. 

 The reference standard working solutions L1, L2, L3, L4, L5 and L6 mg/L were injected onto the 

 HPLC in duplicate and the mean peak area was plotted against concentration (mg/L). The 

 correlation coefficient (r), slope (b) and intercept (a) were calculated for 0 and 24 hours. 

2.6.5 Precision (% RSD) and Accuracy (% Recovery) 

2.6.5.1 Determination of Homogeneity of Mixing and Stability Sample in Dose Formulation 

The test media samples for homogeneity and stability was treated and provided by Toxicology 

Department are as below: 

Treated Samples/Group 

(mg/mL) Replication

Theoretical 

Concentration 

(mg/L)

Theoretical 

Concentration Level 

based on Purity of 

Test Item (mg/L)

Control Level 

(0)

Blank

R1

0.00 0.00

R2

R3

R4

R5

R6

(5 mg/mL)

LR1

5000 4845

LR2

LR3

LR4

LR5

LR6

(100 mg/mL)

HR1

100000 96900

HR2

HR3

HR4

HR5

HR6
JRF Study Number: 228-2-13-5312 (Final Report) Page 16 of 43 

A volume of 0.1 mL from 5 mg/mL solution (LR1, LR2, LR3, LR4, LR5 and LR6) were 

transferred into separate volumetric flasks of 10 mL capacity and the volume of each flask was 

made up to the mark with RO water. The dilution factor was 100. 

A volume of 0.05 mL from 100 mg/mL solution (HR1, HR2, HR3, HR4, HR5 and HR6) were 

transferred into separate volumetric flasks of 100 mL capacity and the volume of each flask was 

made up to the mark with RO water. The dilution factor was 2000. 

The blank RO water in six replication and above prepared samples were injected onto HPLC in 

accordance with section 2.6.2

2.6.6 Calculation

Y - a

Sodium 3-Nitrobenzensulfonate a.i. concentration, (mg/L) = ------ x D 

 b

Where,

Y = Peak area of the sample 

a = Constant

b = Regression co-efficient for Y on „X‟

D = Dilution factor

The % RSD was calculated using the following formula:

 Standard Deviation

Precision (% RSD) = ---------------------------------------- x 100

 Mean Recovered Concentration

The Accuracy (% Recovery) was calculated using the following formula:

Recovered Concentration

% Recovery = -------------------------------- x 100

Fortified Concentration
JRF Study Number: 228-2-13-5312 (Final Report) Page 17 of 43 

3. RESULTS 

3.1 Specificity 

The specificity of the method for the determination of Sodium 3-Nitrobenzensulfonate active 

ingredient concentration in dose formulation was studied by injecting RO water, standard solution 

of Sodium 3-Nitrobenzensulfonate and test item solution. Since there was no interference between 

the peaks of active ingredient standard, sample as well as solvent (acetonitrile) and untreated dose 

formulation, the method was considered to be specific for the analyte.

3.2 Linear Dynamic Range 

The linearity of the method for precision and accuracy was established by injecting six different 

concentrations viz. 10.04, 20.07, 40.14, 60.21, 80.28 and 100.35 mg/L (Refer Table 1) of Sodium 

3-Nitrobenzensulfonate reference standard solution in duplicate and the mean peak area was 

plotted against concentration (mg/L). The correlation coefficient (r) was 0.999 (Refer Figure 1).

The linearity of the method for stability at the level of dose formulation concentration treated and 

provided by the toxicology study was established by injecting the six different concentrations viz. 

10.04, 20.07, 40.14, 60.21, 80.28 and 100.35 mg/L mg/L (Refer Tables 2) of Sodium 3-

Nitrobenzensulfonate reference standard solution in duplicate and the mean peak area was plotted 

against concentration (mg/L). The correlation coefficient (r) was 0.999 (Refer Figures 2).

3.3 Limit of Quantitation 

The limit of quantitation (LOQ) of Sodium 3-Nitrobenzensulfonate was determined by injecting 

different fortified in dose formulation at various concentrations by test item. The minimum signal 

to noise ratio  10:1 was considered for LOQ. The lowest quantifiable concentration (LOQ) of 

Sodium 3-Nitrobenzensulfonate in dose formulation with signal to noise ratio of 14.88 was 0.50

mg/L (Refer Table 3).

3.4 Precision (% RSD) 

Precision of the analytical method was determined by analysing dose formulation fortified with test 

item at the 5 and 100 mg/mL. The precision (% RSD) of analytical method in dose formulation at 5 

mg/mL was 0.07%. The corresponding precision (% RSD) at 100 mg/mL was 0.85% (Refer 

Table 4).
JRF Study Number: 228-2-13-5312 (Final Report) Page 18 of 43 

3.5 Accuracy (% Recovery) 

Accuracy of the analytical method was determined by analysing dose formulation fortified with test 

item at the 5 and 100 mg/mL. The mean accuracy (% recovery) of Sodium 3-Nitrobenzensulfonate 

in dose formulation at 5 mg/mL was 101.98%. The corresponding mean accuracy (% recovery) at 

100 mg/mL was 99.60% (Refer Table 5).

3.6 Homogeneity and Stability 

The homogeneity and stability of test item in dose formulation was determined by analysing dose 

formulation fortified with test item at the level of dose formulation treated and provided by the 

toxicology study at different intervals i.e, 0 h and 24 h. The homogeneity and stability was 

calculated as (%) recovery. The results are summarized in table provided below: 

Interval

5 mg/mL (% Recovery) 100 mg/mL (% Recovery)

Replication 0 h 24 h Replication 0 h 24 h

LR1 101.95 101.88 HR1 99.77 99.94

LR2 102.02 101.69 HR2 100.32 98.07

LR3 101.97 101.77 HR3 100.28 99.98

LR4 102.09 101.88 HR4 98.23 98.42

LR5 101.99 101.89 HR5 98.92 99.91

LR6 101.86 102.11 HR6 100.08 99.37

Mean 101.98 101.87 Mean 99.60 99.28

SD 3.6572 6.9368 SD 821.0439 813.9644

% RSD 0.07 0.14 % RSD 0.85 0.85

 

 
JRF Study Number: 228-2-13-5312 (Final Report) Page 19 of 43 

4. CONCLUSION 

From the results of the validation it is concluded that the method is sensitive and precise for the 

analysis of active ingredient content concentration, homogeneity and stability of Sodium 3-

Nitrobenzensulfonate in dose formulation. The results of validation criteria are within the guidelines 

(U.S. EPA OPPTS 860.1340 and SANCO/3029/99 rev.4) specified limit. 

 
JRF Study Number: 228-2-13-5312 (Final Report) Page 20 of 43 

5. REFERENCES 

European Commission 2000: Residues: Guidance for generating and reporting methods of analysis 

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

(part A, Section 5) of Directive 91/414; SANCO/3029/99 rev. 4 of 11 July 2000,”Directorate 

General Health and Consumer Protection”. 

JRF SOP Number: 184 “Determination of Active Ingredient Content, Stability and Homogeneity of 

Test Item in Test vehicle”. 

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). 

U.S. EPA, 1996: OPPTS 860.1340 “Residue Analytical Method”. United States Environmental 

Protection Agency, Residue Chemistry Test Guidelines, EPA 712-C-96-174 (August 1996). 
JRF Study Number: 228-2-13-5312 (Final Report) Page 21 of 43 

10 30 50 70 90 110

Concentration (mg/L)

400

1100

1800

2500

3200

3900

4600

Mean Peak Area (Thousands)

Study N° 5312 Fig. 1

Linearity Curve of Sodium 3-Nitrobenzensulfonate

Intercept with y-axis (a) = 12827.10

Slope of the line (b) = 40335.43

Correlation co-efficient or 'r' value = 0.999

Equation : Y = bX + a

Y = 40335.43X + 12827.10

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation 

TABLE 1

Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate for Precision (%RSD) and 

Accuracy (% Recovery) and the Level of Test Concentration during Toxicology Study (0 hr) 

Concentration (mg/L) Replication Peak Area Count Mean Peak Area Count % Variation

10.04

R1 411334

410876.5 0.22

R2 410419

20.07

R1 821539

821439.0 0.02

R2 821339

40.14

R1 1652262

1649433.5 0.34

R2 1646605

60.21

R1 2434775

2432567.5 0.18

R2 2430360

80.28

R1 3251485

3250852.5 0.04

R2 3250220

100.35

R1 4058912

4059743.0 0.04

R2 4060574

Typical Calculation

Maximum Area - Minimum Area

% Variation = ------------------------------------------ x 100

Maximum Area

 411334  410419

= ------------------------- x 100

 411334

=0.22%

FIGURE 1 

Linear Dynamic Curve for Sodium 3-Nitrobenzensulfonate for Precision (%RSD) and Accuracy 

(% Recovery) and the Level of Test Concentration during Toxicology Study (0 hr) 
JRF Study Number: 228-2-13-5312 (Final Report) Page 22 of 43 

10 30 50 70 90 110

Concentration (mg/L)

400

1100

1800

2500

3200

3900

4600

Mean Peak Area (Thousands)

Study N° 5312 Fig. 2

Linearity Curve of Sodium 3-Nitrobenzensulfonate

Intercept with y-axis (a) = 9870.53

Slope of the line (b) = 40395.97

Correlation co-efficient or 'r' value = 0.999

Equation : Y = bX + a

Y = 40395.97X + 9870.53

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation 

TABLE 2 

Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate at the Level of 

Test Concentration during Toxicology Study (24 h) 

Concentration (mg/L) Replication Peak Area Count Mean Peak Area Count % Variation

10.04

R1 410777

410674.5 0.05

R2 410572

20.07

R1 817289

818671.0 0.34

R2 820053

40.14

R1 1650343

1647216.0 0.38

R2 1644089

60.21

R1 2433453

2437494.0 0.33

R2 2441535

80.28

R1 3245044

3243800.0 0.08

R2 3242556

100.35

R1 4068055

4068151.0 0.00

R2 4068247

Typical Calculation

Maximum Area - Minimum Area

% Variation = ------------------------------------------ x 100

Maximum Area

 410777  410572

= ------------------------ x 100

 410777

=0.05%

FIGURE 2 

Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate at the Level of 

Test Concentration during Toxicology Study (24 h) 
JRF Study Number: 228-2-13-5312 (Final Report) Page 23 of 43 

 Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation 

TABLE 3 

Limit of Quantitation of Sodium 3-Nitrobenzensulfonate in Dose Formulation 

Solution 

Concentration 

(mg/L)

Replication Peak Area 

Count

Mean Peak 

Area Mean Noise Signal to Noise Ratio (S/N) Remark

0.1

I 3804

3798.0

1298.00

2.93 -

II 3792

0.5

I 19188

19311.5 14.88 LOQ

II 19435

1.0

I 39375

39717.5 30.60 -

II 40060

1.5

I 59004

59110.5 45.54 -

II 59217

Replication Total Area of Noise in Blank 

(a) N° of Noise in Blank (b) Average = a/b

I 1456 1 1456.00

II 1140 1 1140.00

Average Noise Area of Blank 1298.00

Typical Calculation

 Mean Peak Area

Signal to Noise Ratio = -------------------------

 Average Noise Area

Limit of Quantitation

19311.5

 ------------- = 14.88

1298.0

0.5 mg/L
JRF Study Number: 228-2-13-5312 (Final Report) Page 24 of 43 

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation 

TABLE 4 

Calculation of Precision (% RSD) and Accuracy (% Recovery) of the Level of Test Concentration during 

Toxicology Study for Sodium 3-Nitrobenzensulfonate in Dose Formulation (0 hr) 

Treated

Dose

Level

(mg/mL)

Theoretical 

Concentration of Test 

Item

(mg/L)

Active 

Ingredient 

Concentration (mg/L)

Replication

Peak

Area

of

Sample

(Y)

Recovered

(mg/L)

Recovery

(%)

Mean

Concentration

(mg/L)

Mean 

Recovery

(%)

SD %

RSD

Blank

(0.00) - -

R1 000 ND -

- - - -

R2 000 ND -

R3 000 ND -

R4 000 ND -

R5 000 ND -

R6 000 ND -

Lower

(5)

5000 4845

R1 2005161 4939.41 101.95

4940.91 101.98 3.6572 0.07

R2 2006586 4942.95 102.02

R3 2005588 4940.47 101.97

R4 2007838 4946.05 102.09

R5 2005995 4941.48 101.99

R6 2003430 4935.12 101.86

Higher

(100)

100000 96900

R1 1962617 96678.77 99.77

96513.59 99.60 821.0439 0.85

R2 1973416 97214.23 100.32

R3 1972534 97170.5 100.28

R4 1932521 95186.48 98.23

R5 1945900 95849.87 98.92

R6 1968726 96981.68 100.08

Typical Calculation

Intercept with y-axis (a)* 12827.10 Slope of the line (b)* 40335.43

Correlation of coefficient (r) 0.999 Purity of Test Item (%) 96.90

Dilution Factor = 100 for (L) and 2000 for (H)

Concentration (mg/L) % Recovery % RSD

Y – a

= ------- x D

b

2005161 – 12827.10

= ----------------------- x 100

40335.43

= 4939.41 mg/L

Quantity Recovered

= ------------------------ x 100

Quantity Fortified

4939.41

= ---------- x 100

4845

= 101.95%

Standard Deviation

= ------------------------- x 100

Mean Concentration

3.6572

= ----------- x 100

4940.91

= 0.07%

Key: * = Refer figure 1 page N° 21 

ND = Non-Detectable (< Limit of Quantitation i.e.,0.5 mg/L) 

 
JRF Study Number: 228-2-13-5312 (Final Report) Page 25 of 43 

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation 

TABLE 5 

Calculation of (%) Recovery and Stability at the Level of 

Test Concentration during Toxicology Study for Sodium 3-Nitrobenzensulfonate in

Dose Formulation (24h) 

Treated

Dose

Level

(mg/mL)

Theoretical 

Concentration of Test 

Item

(mg/L)

Active 

Ingredient 

Concentration (mg/L)

Replication

Peak

Area

of

Sample

(Y)

Recovered

(mg/L)

Recovery

(%)

Mean

Concentration

(mg/L)

Mean 

Recovery

(%)

SD %

RSD

Blank

(0.00) - -

R1 000 ND -

- - - -

R2 000 ND -

R3 000 ND -

R4 000 ND -

R5 000 ND -

R6 000 ND -

Lower

(5)

5000 4845

R1 2003930 4936.28 101.88

4935.62 101.87 6.9368 0.14

R2 2000199 4927.05 101.69

R3 2001602 4930.52 101.77

R4 2003847 4936.08 101.88

R5 2003963 4936.36 101.89

R6 2008439 4947.45 102.11

Higher

(100)

100000 96900

R1 1965804 96838.05 99.94

96203.24 99.28 813.9644 0.85

R2 1929368 95034.11 98.07

R3 1966690 96881.92 99.98

R4 1936051 95364.98 98.42

R5 1965311 96813.64 99.91

R6 1954668 96286.71 99.37

Key: * = Refer figure 2 page N° 22 

ND = Non-Detectable (< Limit of Quantitation i.e.,0.5 mg/L) 

 Dilution Factor = 100 for (L) and 2000 for (H) 
JRF Study Number: 228-2-13-5312 (Final Report) Page 26 of 43 

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation 

APPENDIX 1 

Chromatograms 
JRF Study Number: 228-2-13-5312 (Final Report) Page 27 of 43 

APPENDIX 1 (Continued) 
JRF Study Number: 228-2-13-5312 (Final Report) Page 28 of 43 

APPENDIX 1 (Continued) 
JRF Study Number: 228-2-13-5312 (Final Report) Page 29 of 43 

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation

APPENDIX 2 

Signed Study Plan and Study Plan Amendment 

JRF Study Number: 228-2-12-5312 Page 1 of 10

STUDY PLAN

VALIDATION OF ANALYTICAL METHOD FOR DETERMINATION OF SODIUM 3-

NITROBENZENSULFONATE ACTIVE INGREDIENT CONCENTRATION IN DOSE FORMULATION

TEST ITEM: SODIUM 3-NITROBENZENSULFONATE

GUIDELINES: SANCO/3029/99 rev.4 AND U.S. EPA OPPTS 860.1340 

SPONSOR 

TIARCO CHEMICAL 

1300 TIACRO DRIVE 

DALTON, GA 30720 

STUDY DIRECTOR: ASFAK SHRIMALI 

TEST FACILITY 

JAI RESEARCH FOUNDATION 

DEPARTMENT OF CHEMISTRY 

VALVADA - 396 108 

DIST. VALSAD 

GUJARAT 

INDIA 

AUGUST - 2012
JRF Study Number: 228-2-13-5312 (Final Report) Page 30 of 43 

APPENDIX 2 (Continued) 

JRF Study Number: 228-2-12-5312 Page 2 of 10

CONTENTS 

PAGE N°

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

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

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

 1.1 Study Director ........................................................................................................................................ 3 

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

 1.3 Study Schedule ....................................................................................................................................... 3 

 1.4 Study Plan and Amendment (if any) Distribution .................................................................................. 3 

2. INTRODUCTION ............................................................................................................................................ 3 

 2.1 Objective ................................................................................................................................................ 3 

 2.2 Regulatory Guidelines ............................................................................................................................ 3 

 2.3 Test Item ................................................................................................................................................ 4 

 2.4 Reference Standard ................................................................................................................................ 5 

3. GOOD LABORATORY PRACTICE (GLP) ................................................................................................... 5 

 3.1 GLP Compliance .................................................................................................................................... 5 

 3.2 Standard Operating Procedures (SOPs) ................................................................................................. 5 

 3.3 Amendment to Study Plan ..................................................................................................................... 5 

 3.4 Deviation from the Study Plan ............................................................................................................... 6 

 3.5 Quality Assurance .................................................................................................................................. 6 

4. EXPERIMENTAL PROCEDURE .................................................................................................................. 6 

 4.1 Equipment/Instruments ......................................................................................................................... 6 

 4.2 Solvents and Chemicals ........................................................................................................................ 6 

 4.3 Validation of the Analytical Method for Sodium 3-Nitrobenzensulfonate by HPLC ........................... 6 

 4.3.1 Specificity ............................................................................................................................... 6 

 4.3.2 Linear Dynamic Range (LDR) ............................................................................................... 7 

 4.3.3 Limit of Quantitation (LOQ) .................................................................................................. 7 

 4.3.4 Precision (% RSD) and Accuracy (% Recovery) from Test Vehicle ...................................... 7 

 4.3.4.1 Calculation of % Recovery (Accuracy) .................................................................... 7 

 4.3.4.2 Calculation of Precision (% RSD) ............................................................................ 7 

 4.3.5 Instrumental Parameters ......................................................................................................... 8 

 4.3.6 Calculation of Active Ingredient Concentration ..................................................................... 8 

 4.3.7 Sample Analyses for Determination of Homogeneity and Stability ....................................... 8 

5. REPORT .......................................................................................................................................................... 8 

6. ARCHIVES ..................................................................................................................................................... 9 

7. REFERENCES ................................................................................................................................................ 9 

8. STUDY PLAN APPROVAL ......................................................................................................................... 10
JRF Study Number: 228-2-13-5312 (Final Report) Page 31 of 43 

APPENDIX 2 (Continued) 

JRF Study Number: 228-2-12-5312 Page 3 of 10

1. GENERAL INFORMATION

1.1 Study Director : Asfak Shrimali

Deputy Study Director : Ashok S. Amruskar

1.2 Test Facility Management : Hemant Desai

1.3 Study Schedule

Study Initiation Date : August 25, 2012

Experiment Start Date : August 30, 2012

Experiment Completion : Latest by August 2012

Draft Report Submission : Latest by September 2012

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

final draft report from the Sponsor.

1.4 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 validate the analytical method for determination of active ingredient 

concentration of Sodium 3-Nitrobenzensulfonate in the dose formulation. 

2.2 Regulatory Guidelines

European Commission 2000: Residues: Guidance for generating and reporting methods of analysis in support 

of pre-registration data requirements for Annex II (part A, Section 4), and Annex III (part A, Section 5) of 

Directive 91/414; SANCO/3029/99 rev. 4 of 11 July 2000, “Directorate General Health and Consumer 

Protection”.

U.S. EPA, 1996: OPPTS 860.1340 “Residue Analytical Method”. United States Environmental Protection 

Agency, Residue Chemistry Test Guidelines, EPA 712-C-96-174 (August 1996). 
JRF Study Number: 228-2-13-5312 (Final Report) Page 32 of 43 

APPENDIX 2 (Continued) 

JRF Study Number: 228-2-12-5312 Page 4 of 10

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

CA Name

CAS Number

Batch/Lot N°

Concentration / Purity

Supplied by 

Manufactured by

Date of Manufacture

Date of Expiry

Appearance / Colour 

Sodium 3-Nitrobenzensulfonate 

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)

Reservol LP

Tiarco Chemical

August 09, 2011

August 09, 2013

Yellow to tan granular solid

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 in room temperature (40 to 90 °F) in the 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: 228-2-13-5312 (Final Report) Page 33 of 43 

APPENDIX 2 (Continued) 

JRF Study Number: 228-2-12-5312 Page 5 of 10

2.4 Reference Standard 

Reference Standard

C.A. Name 

Molecular Weight

Molecular Formula

CAS N°

LOT N°

Date of Receipt

Date of Expiry

Purity 

Source

Storage Condition 

JRF Entry Number

Sodium 3-Nitrobenzensulfonate 

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%

ALDRICH

Room Temperature

CPC-54

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 Economic Co-operation 

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

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. 
JRF Study Number: 228-2-13-5312 (Final Report) Page 34 of 43 

APPENDIX 2 (Continued) 

JRF Study Number: 228-2-12-5312 Page 6 of 10

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 47582). 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 

Analytical Balance, HPLC and Refrigerator. 

4.2 Solvents and Chemicals 

Acetonitrile, formic acid, RO water and Milli-Q Water. 

4.3 Validation of the Analytical Method for Sodium 3-Nitrobenzensulfonate by HPLC 

The analytical method for the determination of Sodium 3-Nitrobenzensulfonate concentration in dose 

formulation will be validated by analysis of test item and blank test vehicle. The validation will cover the 

aspects namely (i) specificity, (ii) linear dynamic range (LDR), (iii) limit of quantitation (LOQ) (iv), precision 

(%, RSD) and accuracy (%, Recovery) from test vehicle (RO Water).

4.3.1 Specificity 

The test item solution, reference solution and blank test vehicle (RO Water) and injected onto a HPLC using 

selected parameters. The interference (if any), in the determination of active ingredient in test item will be 

reported. Interference should not exceed 30% peak area of the target active ingredient at LOQ level. 
JRF Study Number: 228-2-13-5312 (Final Report) Page 35 of 43 

APPENDIX 2 (Continued) 

JRF Study Number: 228-2-12-5312 Page 7 of 10

4.3.2 Linear Dynamic Range (LDR) 

Six different concentrations of Sodium 3-Nitrobenzensulfonate will be prepared and injected onto HPLC in two 

replicates. The linear calibration curve will be established by plotting the mean peak area against concentration 

(mg/L). The correlation coefficient (r), intercept (a) and slope (b) will be calculated. The regression equation 

will be established. 

4.3.3 Limit of Quantitation (LOQ) 

The minimum quantity of test item (Sodium 3-Nitrobenzensulfonate ) fortified in the test vehicle, which could be 

produced a signal to noise ratio  10:1, will be considered as limit of quantitation (LOQ). 

4.3.4 Precision (% RSD) and Accuracy (% Recovery) from Test Vehicle 

Precision and accuracy will be established at the level of test concentration used during the toxicology study. 

Six determinations will be made at each fortification level. The sample will be diluted, if necessary and 

analysed by the HPLC. 

The active ingredient concentration in each replicate, mean of active ingredient concentration, SD and % RSD 

will be calculated and reported. Each replicate of sample solution will be injected in duplicate. The test item 

will be fortified in test vehicle will be injected onto HPLC in accordance with the parameters described in 

section 4.3.5. 

4.3.4.1 Calculation of % Recovery (Accuracy)

 Recovered Concentration 

% Recovery (Accuracy) = -------------------------------- x 100 

Fortified Concentration 

Accuracy/recovery values should fall within 70 to 110%. If the majority of recoveries do not fall within the 

expected range the reason will be identified and corrections will be made to the method and if required, the 

validation will be repeated. 

4.3.4.2 Calculation of Precision (% RSD)

Precision will be defined by the relative standard deviation (% RSD). The % RSD will be calculated at each 

fortification level. The %RSD should be  20% at each level of fortification. The precision (% RSD) will be 

calculated as follows: 

 Standard deviation 

Precision (% RSD) = --------------------------------------------- x 100 

 Mean Recovered Concentration 
JRF Study Number: 228-2-13-5312 (Final Report) Page 36 of 43 

APPENDIX 2 (Continued) 

JRF Study Number: 228-2-12-5312 Page 8 of 10

4.3.5 Instrumental Parameters [Reference JRF Research Number: RES-2-12-5315]

Instrument : HPLC

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

Mobile Phase : Acetonitrile : 0.1% Formic acid in Water (70 : 30) v/v

Wave Length : 260 nm

Flow Rate : 1.0 mL/minute

Injection Volume : 20 μL

4.3.6 Calculation of Active Ingredient Concentration

Y = bX + a 

Y - a

A.I. Concentration (X) = -------- x D 

b

Where,

Y = Peak area of the sample

a = Constant

b = Regression Coefficient for Y on X

D = Dilution factor (if applicable)

4.3.7 Sample Analyses for Determination of Homogeneity and Stability

The dose formulation samples for homogeneity and stability will be prepared and provided by Toxicology 

Department at two concentration levels along with control according to the procedure described in standard 

operating procedure JRF/TOX/288. All the samples will be stored at room temperature during analyses. For the 

stability, the samples will be analysed at the interval of 0 and 24 hrs.

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

- Complete details of the experimental procedure

- 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)
JRF Study Number: 228-2-13-5312 (Final Report) Page 37 of 43 

APPENDIX 2 (Continued) 

JRF Study Number: 228-2-12-5312 Page 9 of 10

6. ARCHIVES 

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

documentation, chromatograms, 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 2000: Residues: Guidance for generating and reporting methods of analysis in support 

of pre-registration data requirements for Annex II (part A, Section 4), and Annex III (part A, Section 5) of 

Directive 91/414; SANCO/3029/99 rev. 4 of 11 July 2000. "Directorate General Health and Consumer 

Protection". 

JRF SOP Number: 184 “Validation of Method and Determination of Active Ingredient Concentration, Stability 

and Homogeneity of Test Item in Dose Formulation/Test Media”.

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). 

U.S. EPA, 1996: OPPTS 860.1340 "Residue Analytical Method". United States Environmental Protection 

Agency, Residue Chemistry Test Guidelines, EPA 712-C-96-174 (August 1996). 
JRF Study Number: 228-2-13-5312 (Final Report) Page 38 of 43 

APPENDIX 2 (Continued) 
JRF Study Number: 228-2-13-5312 (Final Report) Page 39 of 43 

APPENDIX 2 (Continued) 
JRF Study Number: 228-2-13-5312 (Final Report) Page 40 of 43 

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation 

APPENDIX 3 

Certificate of Analysis of Test Item
JRF Study Number: 228-2-13-5312 (Final Report) Page 41 of 43 

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation

APPENDIX 4 

Certificate of Analysis of Reference Standard of Sodium 3-Nitrobenzensulfonate
JRF Study Number: 228-2-13-5312 (Final Report) Page 42 of 43 

Validation of Analytical Method for Determination of Active Ingredient Concentration of 

Sodium 3-Nitrobenzensulfonate in Dose Formulation 

APPENDIX 5 

GLP Endorsement Compliance 
JRF Study Number: 228-2-13-5312 (Final Report) Page 43 of 43 

APPENDIX 5 (Continued)