## FINAL REPORT

Study Title



## DEVELOPMENT AND VALI DATION OF

AN ANALYTICAL METHOD FOR THE ANALYSIS OF

1

- PROPANESULFONIC ACID , 2

- HYDROXY

- 3

- (2

- PROPEN

- 1

-

YLOXY)

- , SODIUM SALT (1:1) IN WATER

Author

M.J.C. Brekelmans, MSc

.

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 500674

Substance 204240 / A

- Page 1 of 15

-1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 2 - 1. CONTENTS

1. CONTENTS

................................................................................................ ...................................... 2

2. STATEMENT OF GLP COM PLIANCE

............................................................................................. 3

3. QUALITY ASSURANCE ST ATEMENT

............................................................................................ 4

4. SUMMARY

................................................................................................ ........................................ 5

5. INTRODUCTION

.............................................................................................................................. 6

5.1. Preface

................................................................................................ ................................... 6

5.2. Aim of the study

...................................................................................................................... 6

5.3. Storage and retention of records and materials

..................................................................... 6

6. MATERIALS

................................................................................................ ...................................... 7

6.1. Test substance

....................................................................................................................... 7

6.2. Elect ronic data capture

........................................................................................................... 7

6.3. List of deviations

..................................................................................................................... 7

7. VALIDATION OF AN ANA LYTICAL METHOD

................................................................................. 8

7.1. Guideline

................................................................................................ ................................ .8

7.2. Reagents

................................................................................................ ................................ 8

7.3. Performance of the study

....................................................................................................... 8

7.4. Analytical method

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

7.5. Formulas

.............................................................................................................................. 10

7.6. Results

................................................................................................ ................................ . 11

7.7. Conclusion

........................................................................................................................... 13

7.8. Figures

................................................................................................ ................................ . 14

TABLES

Table 1 Statistical parameters of the calibration curve

.................................................................. 11

Table 2 Accuracy samples

............................................................................................................. 11

Table 3 Stability of the analytical system and end solutions

.......................................................... 12

Table 4 Stored accuracy samples

.................................................................................................. 12

FIGURES

Figure 1 HPLC

- RI chromatogram of a 156 mg/l test substance solution [res. id. 1657].

................ 14

Figure 2 HPLC

- RI chromatogram of the blank accuracy sample [res. id. 1646].

............................ 14

Figure 3 Regression line: response of the test substance as a function of concentration [cal.

curve id. 1626 ].

.................................................................................................................. 14

APPENDICES

Appendix 1 Certificate of analysis

........................................................................................................ 151

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 5

- 4. SUMMARY

The analytical method developed for the quantitative analysis of the test substance in water was

successfully validated for the following parameters:

- Specificity

- Linearity

- Accuracy and repeatability

- Limit of quantification (LOQ)

- Stability of the analytical system and end solutions

- Stability of stock solutions

- Storage stability of samples1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 6

- 5. INTRODUCTION

5.1. Preface

Sponsor Society of Chemical Manufacturers and Affiliates

1850 M Street, NW Suite 700

WASHINGTON, DC 20036

USA

Study Monitor Dr. C. T. Helmes

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director M.J.C. Brekelmans , MSc

.

Study Plan Start : 07 M arch 201 3

Completion : 03 M ay 201 3

5.2. Aim of the study

The aim of the study was to develop and to validate an analytical method for the quantitative analysis

of 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) in water.

5.3. Storage and retention of records and materials

Records and materials pertaining to the study including protocol, raw data, specimens (except

specimens requiring refrigeration or freezing) and the final report are retained in the WIL Research

Europe archive s for a period of at least 2 years after finalization of the report. After this period, the

sponsor will be contacted to determine how the records and materials should be handled. WIL

Research Europe will retain information concerning decisions made.

Thos e specimens requiring refrigeration or freezing will be retained by WIL Research Europe for as

long as the quality of the specimens permits evaluation but no longer than three months after

finalization of the report.

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than

10 years after finalization of the report. After this period the sample will be destroyed.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 7 - 6. MATERIALS

6.1. Test substance

6.1.1. Test substance information

Identification 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1)

Synonyms COPS_1 or CS

- AHPS

Structure

O SO 3 -

OH Na +

Molecular formula C 6 H 11 NaO 5 S

Molecular weight 218.22

CAS Number 52556

- 42

- 0

Description Clear colourless liquid

Batch 12E1101

Purity/Composition

Sodium -1- allyloxy -2-

hydroxypropane sulphonate

3- allyl

- 1,2

- propanediol

Water CAS Number Percent

52556

- 42

- 0 38.64

123

- 34

- 2 2.26

7732

- 18

- 5 59.1

Test substance storage At room temperature in the dark

Stability under storage conditions Stable

Expiry date 13 August 2013

The certificate of analysis is shown in Appendix 1

.

6.1.2. Study specific test substance information

Purity/composition correction factor

required No

6.2. Electronic data capture

System control, data acquisition and data processing were performed using the following programme:

- Empower version 7 .00 (Waters, Milford, MA, USA)

Temperature, relative humidity and/or atmospheric pressure during sample storage and/or

performance of the studies w as monitored continuously using the following programme:

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,

USA)

6.3. List of deviations

6.3.1. List of protocol deviations

Mean accuracy of the 100 mg/l accuracy samples stored in the freezer was 111% which is outside the

criterion range of 70

– 110%. Since this is only slightly outside the range and since mean accuracy of

the stored 1000 mg/l accuracy samples was 102%, it was still concluded that the samples were stable

during storage in the freezer. The study integrity was not adversely affected by this deviation

.

6.3.2. List of standard operating procedures deviations

There were no deviations from standard operating procedures (SO Ps).1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 8

- 7. VALIDATION OF AN ANA LYTICAL METHOD

7.1. Guideline

The study was based on the following guideline:

European Commission: Guidance for Generating and Reporting Methods of Analysis in Support of

Pre

- registration Data Requirements for Annex II (Part A, sect ion 4) and Annex III (Part A, section 5) of

Directive 91/414, SANCO/3029/99 rev. 4 (11/07/00).

7.2. Reagents

Water Tap water purified by a Milli

- Q water purification

system (Millipore, Bedford, MA, USA)

Di

- So dium hydrogenphosphate dihydrate Merck, Darmstadt, Germany

( Na 2 H2PO4 · 2H 2 O)

Sodium dihydrogenphosphate di hydrate Merck

(NaH 2 PO 4 · 2H 2 O)

Sodium nitrate Merck

(NaNO 3 )

All reagents were of analytical grade, unless specified otherwise.

7.3. Performance of the study

A high performance liq uid chromatographic method with refractive index (RI) detection for quantitative

analysis of the test substance in water was developed. Validation of the analytical method was

performed for the following parameters:

Specificity

A test substance solution a nd blank accuracy sample were analysed by single injection. The analytical

method was found to be specific if the blank chromatogram showed no response for the test

substance or a response of < 30% of the limit of quantification.

Linearity

Calibration solutions were analysed in duplicate. The response of the calibration solutions was

correlated with concentration using regression analysis with a 1/concentration 2 weighting factor. A

calibration curve with a coefficient of correlation (r) of > 0.99 and ba ck calculated accuracies of the

calibration solutions in the range 85

- 115% was accepted.

Accuracy and repeatability

Accuracy samples were analysed by single injection into the analytical system. The analytical method

was considered applicable for the det ermination of the test substance if the mean accuracy was in the

range 70

- 110% and the coefficient of variation was ≤ 20%.

Limit of quantification

The limit of quantification (LOQ) is defined as the lowest concentration level at which an accuracy in

the range 70

- 110% and a repeatability of ≤ 20% is demonstrated. The LOQ was obtained from the

data of the accuracy

- and repeatability test

.1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 9

- Stability of the analytical system and end solutions

Calibration solutions were injected throughout the validation se quence including the beginning and

end. The analytical system and/or end solutions were found to be stable if the coefficient of variation

on the responses of the solutions was ≤ 20%

.

Stability of stock solutions

Stock solutions of the test substance were stored at room temperature for at least 12 hours. Additional

calibration solutions were prepared and analysed by single injection. The stock solutions were found to

be stable if the coefficient of variation on the response factors of the calibration solut ions prepared with

fresh and stored stock solutions was ≤ 10%.

Storage stability of samples

Additional accuracy samples were prepared and stored in the freezer ( ≤

- 15°C ) for at least 16 hours.

On the day of analysis, the samples were defrosted at room te mperature and treated identically as

freshly analysed accuracy samples. The stored samples were found to be stable if the mean accuracy

was in the range 70

- 110%.

7.4. Analytical method

7.4.1. Analytical conditions

Instrument Alliance Separation Module 2695 (Waters, M ilford, MA, USA)

Detector Refractive Index Detector 2414 (Waters)

Column

Stationary phase Synergi Hydro

- RP 80A

Dimensions 250 mm × 4.6 mm i.d., dp = 4 μm

Brand Phenomenex

, Torrance, CA , USA

Column temperature 40°C ± 1°C

Injection volume 50 μl

Mobile phase water containing 0.05M Na 2 HPO 4 , 0.05M NaH 2 PO 4 and 0.05M

NaNO 3

Flow 1 ml/min

RI detection

Temperature 30°C

Sensitivity 4

Time constant 1 seconds

7.4.2. Preparation of solutions

Stock and spiking solutions

Stock solutions of the test substance were prepared in water at concentrations of 5530

- 6660 mg/l.

Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of

the spiking solutions was water.

Calibration solut ions

Five calibration solutions in the concentration range of 80

– 1200 mg/l were prepared from two stock

solutions. The end solution of the calibration solutions was mobile phase

.

Accuracy samples

W ater was spiked with the test substance at a target concentration of 100 or 100 0 mg/l. The accuracy

samples were analysed without further treatment

.

The blank accuracy sample was prepared and treated similarly to the accuracy samples.1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 10

- 7.5. Formulas

Respons e (R) Peak area of the test substance [units]

Response factor (R f )

N f C R R

=

where:

C N = nominal concentration [mg/l]

Calibration curve b C a R N +

=

where:

a = slope [units × l/mg]

b

= intercept [units]

Analysed concentration (C A ) [mg/l] d

a b) (R C A × −

=

where:

d

= dilution factor

Accuracy [%] 100 C C

N

A

×1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 11

- 7.6. Results

7.6.1. Specificity

Chromatograms of a test substance solution and the blank accuracy sample are shown in Figure 1

and Figure 2

, respectively.

The chromatogram of the test substance solution showed one test substance peak. The area of this

peak was used as response in the calculations.

The chromatogram of the blank accuracy sample showed no peak at the retention time of the test

substance. Since no interferences were detected, the specificity requirements were met and the

analytical method was found to be specific for the test substance.

7.6.2. Linearity

The calibration line was constructed using all data points

. Figure 3 illustrates the calibration curve and

Table 1 shows the statistical parameters. There was a linear re lationship between response and test

substance concentration in the range of 79.9

– 1200 mg/l (in end solution). Since the coefficient of

correlation (r) was > 0.99 and the back calculated accuracies of the data points were in the range

85

- 115% the calibration line was accepted.

Table 1 Statistical parameters of the calibration curve

Slope 6. 8 0 × 10 1

Intercept

- 1.55 × 10 2

Weighting factor 1/concentration 2

r 0.99993

7.6.3. Accuracy and repeatability

The results of the accuracy samples are given in Table 2

. Since the mean accuracy at each

concentration level fell in the criterion 70

- 110% and the coefficient of variation was ≤ 20% the

analytical method was accepted for the analysis of the test substance in water in the target

concentration range of 100

- 1000 mg/l.

Table 2 Accuracy samples

Target

concentration

[mg/l] Nominal

concentration

[mg/l] Analysed

concentration

[mg/l] Accuracy

[%] Mean

accuracy

[%] Coefficient of

variation

[%]

100 102 110 108 106 1.8

102 109 107

102 109 107

102 106 104

102 106 103

1000 1020 1039 102 104 1.3

1020 1073 105

1020 1072 105

1020 1071 105

1020 1060 1041- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 12

- 7.6.4. Limit of quantification

The limit of quantification (LOQ) was assessed at 100 mg/l in water.

7.6.5. Stability of the analytical system and end solutions

The results of the stability of the analytical system and end solutions are given in Table 3

. Since the

coefficient of variation at all concentration levels was ≤ 20% the analytical system and end solutions

were stable over at least an 18.1 hour time interval.

Table 3 S tability of the analytical system and end solutions

Nominal concentration

[mg/l] Elapsed time

[hours] Coefficient of variation

[%]

79.9 19. 5 1.7 (n= 8 )

15 6 19. 5 1.5 (n= 8 )

1200 18. 1 1.3 (n=8)

7.6.6. Stability of stock solutions

The coefficient of variation on the response factors of the calibration solutions prepared with fresh and

stored stock solutions was 2 .1% . Since the value was ≤ 10% the stock solutions were stable when

stored at room temperature for at least 1 day.

7.6.7. Storage stability of samples

Mean accuracy of the 100 mg/l accuracy samples stored in the freezer was 111% which is outside the

criterion range of 70

– 110%. Since this is only slightly outside the range and since mean accuracy of

the stored 1000 mg/l accuracy samples was 102%, it was still concluded that the samples were stable

during storage in the freezer.

Table 4 Stor ed accuracy samples

Target

concentration

[mg/l] Nominal

concentration

[mg/l] Analysed

concentration

[mg/l] Accuracy

[%] Mean accuracy

[%]

100 104 121 117 111

104 110 106

1000 1035 1050 101 102

1035 1056 1021

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 13

- 7.7. Conclusion

The analytical method was validated for the following parameters:

Specificity specific

Linearity r = 0.99993

Accuracy 106 and 104 %

Repeatability 1.8 and 1.3 %

Limit of quantification 100 mg/l

Stability analytical system and end solutions stable

Stability s tock solutions stable

Storage stability stable1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 14

- 7.8. Figures

Figure 1 HPLC

- RI chromatogram of a 156 mg/l test substance solution [res. id. 1657 ].

Figure 2 HPLC

- RI chromatogram of the blank accuracy sample [res. id. 1646 ].

Figure 3 Regression line: response of the test substance as a function of concentration [cal.

curve id. 1626 ].TS204240 - 13.253MV

-5.50 -5.00 -4.50 -4.00 -3.50 -3.00 -2.50 -2.00 -1.50 -1.00 -0.50 0.00 0.50

Minutes 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00MV

-550.00 -500.00 -450.00 -400.00 -350.00 -300.00 -250.00 -200.00 -150.00 -100.00 -50.00 0.00

Minutes 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00Area

-10000.0 0.0 10000.0 20000.0 30000.0 40000.0 50000.0 60000.0 70000.0 80000.0 90000.0

mg/l 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00 550.00 600.00 650.00 700.00 750.00 800.00 850.00 900.00 950.00 1000.00 1050.00 1100.00 1150.00 1200.001

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Project 500674

Final report

- Page 15

- Appendix 1 Certificate of analysis