FINAL REPORT

Study Title

DEVELOPMENT AND VALIDATION 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

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1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) Project 500674

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1. CONTENTS

1. CONTENTS ......................................................................................................................................2

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

3. QUALITY ASSURANCE STATEMENT ............................................................................................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. Electronic data capture...........................................................................................................7

6.3. List of deviations .....................................................................................................................7

7. VALIDATION OF AN ANALYTICAL 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 ........................................................................................................ 15


1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) Project 500674

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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 samples
1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) Project 500674

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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 March 2013

Completion : 03 May 2013

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 archives 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.

Those 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

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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 SO3

-

OH

Na+

Molecular formula C6H11NaO5S

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 was 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 (SOPs).
1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) Project 500674

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7. VALIDATION OF AN ANALYTICAL 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, section 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-Sodium hydrogenphosphate dihydrate Merck, Darmstadt, Germany

(Na2H2PO4 · 2H2O)

Sodium dihydrogenphosphate dihydrate Merck

(NaH2PO4 · 2H2O)

Sodium nitrate Merck

(NaNO3)

All reagents were of analytical grade, unless specified otherwise.

7.3. Performance of the study

A high performance liquid 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 and 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/concentration2 weighting factor. A 

calibration curve with a coefficient of correlation (r) of > 0.99 and back 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 determination 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.
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Stability of the analytical system and end solutions

Calibration solutions were injected throughout the validation sequence 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 solutions 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 temperature 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, Milford, 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 Na2HPO4, 0.05M NaH2PO4 and 0.05M 

NaNO3

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 solutions

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 

Water was spiked with the test substance at a target concentration of 100 or 1000 mg/l. The accuracy 

samples were analysed without further treatment.

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

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

Response factor (Rf)

N

f C

R R =

where:

CN = nominal concentration [mg/l]

Calibration curve R = a CN + b

where:

a = slope [units × l/mg]

b = intercept [units]

Analysed concentration (CA) d [mg/l]

a

(R b) CA × − =

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

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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 relationship 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 6.80 × 101

Intercept -1.55 × 102

Weighting factor 1/concentration2

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 104
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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 Stability 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)

156 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 Stored 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 102
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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 stock solutions stable

Storage stability stable
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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 - 13.253

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Appendix 1 Certificate of analysis