### Endpoint study record: Analytical methods. HPLC-RI. WIL Research Project 500674

file:///C|/

... net Files/Content.Outlook/6RSURWTF/Endpoint study record Analytical methods_ HPLC-RI_ WIL Research Project 500674.html [9/13/2013 2:27:56 PM] Printing Date 2013-06-12 12:19:03 CEST Restriction of specific regulatory purposes

Confidentiality

Owner (TS 204240) 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) / WIL Research Europe B.V. / 's-Hertogenbosch / Netherlands Legal entity
owner WIL Research Europe B.V. / 's-Hertogenbosch / Netherlands 

# Endpoint study record: Analytical methods. HPLC-RI. WIL Research Project 500674

UUID IUC5-e39aad31-4419-4cd5-a046-7ea12223d61e Dossier UUID 0 Author TRJ / WIL Research Europe B.V. / 's-Hertogenbosch / Netherlands Date 2013-06-12 12:18:54 CEST Remarks



# Administrative Data
Purpose flag key study; robust study summary Study result type experimental result Study period 07 March 2013 - 03 May 2013 Reliability 1 (reliable without restriction) Rationale for reliability incl. deficiencies The study has been performed according to EC recommendations and in compliance with the principles of GLP. 

# Data source Background information
A high performance liquid chromatographic method with refractive index (RI) detection was validated for the quantitative analysis of the substance in water. The validation included the determination of the following parameters: - Specificity - Linearity - Accuracy and repeatability - Limit of quantification - Stability of the analytical system and end solutions - Stability of stock solutions - Storage stability of samples 

### Reference

### Endpoint study record: Analytical methods. HPLC-RI. WIL Research Project 500674

file:///C|/

... net Files/Content.Outlook/6RSURWTF/Endpoint study record Analytical methods_ HPLC-RI_ WIL Research Project 500674.html [9/13/2013 2:27:56 PM] Reference type Author Year Title Bibliographic
source Testing laboratory Report
no. Owner company Company study no. Report date study report Brekelmans, MJC 2013 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 WIL Research Europe B.V., Hambakenwetering7, 5231 DD ‘sHertogenbosch, The Netherlands 500674 Society of Chemical Manufacturers and Affiliates, 1850 M Street, NW Suite 700, WASHINGTON, DC 20036, USA 2013- 06-10



### Data protection claimed

yes 

# Materials and methods
Matrix / medium

water 

### Test guideline

Qualifier Guideline Deviations according to other 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). no



### GLP compliance

yes 

## Test materials Identity of test material same as for substance defined in section 1 (if not read-across)

yes 

### Test material identity

Identifier Identity CAS number 52556-42-0 

### Test material form
other: liquid 

### Details on test material
- Name of test material (as cited in study report): 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) - Description: Clear colourless liquid - Storage condition of test material: At room temperature in the dark 

### Confidential details on test material
- Purity/Composition: 38.64% of CAS# 52556-42-0, 2.26% of CAS# 123-34-2, 59.1% of water - Lot/batch No.: 12E1101 - Expiration date of the lot/batch: 13 August 2013 

## Principles of analytical methods Instrument / detector

other: HPLC-RI 

### Details on analytical method
Instrument: Alliance Separation Module 2695 (Waters, USA) Detector: Refractive Index Detector 2414 (Waters) Column: Synergi Hydro-RP 80A, 250 mm x 4.6 mm i.d., dp =4 μm (Phenomenex, 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

### Endpoint study record: Analytical methods. HPLC-RI. WIL Research Project 500674

file:///C|/

... net Files/Content.Outlook/6RSURWTF/Endpoint study record Analytical methods_ HPLC-RI_ WIL Research Project 500674.html [9/13/2013 2:27:56 PM] Flow: 1 mL/min RI detection: 30°C; sensitivity of 4; time constant of 1 seconds
Stock solutions of the substance in water: 5530 - 6660 mg/L (nominal) Five calibration solutions (80 – 1200 mg/L nominal) in mobile phase: prepared from two stock solutions
Chromatogram: one test substance peak (see attached document). Response: peak area of the test substance.


## Confirmatory method (if applicable) Any other information on materials and methods incl. tables

SPECIFICITY 

### Single injection of test substance solution and a blank accuracy sample. If a peak at the retention time of the test substance was present in the blank chromatogram, the response was compared with the limit of quantification.

LINEARITY Duplicate analysis of five calibration solutions. The responses were plotted against the nominal concentrations using regression analysis (linear least squares method) with a 1/concentration^2 weighting factor.

ACCURACY AND REPEATABILITY Matrix: tap water purified by a water purification system (Millipore, USA) Spiking solutions in water: (prepared from) a test substance stock solution in water. Accuracy samples: Water was spiked with 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. Accuracy samples were analysed by single injection.

LIMIT OF QUANTIFICATION (LOQ) LOQ is the lowest concentration level at which an accuracy in the range 70-110% and a repeatability of = 20% was demonstrated and was obtained from the data of the accuracy- and repeatability test.

STABILITY OF THE ANALYTICAL SYSTEM AND END SOLUTIONS Each of three calibration solutions was injected throughout the validation sequence including the beginning and end.

STABILITY OF STOCK SOLUTIONS Single injection of calibration solutions prepared from two fresh and two stored stock solutions. Storage conditions: at room temperature for at least 12 hours.

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.


# Results and discussions Recovery results and characteristics of analytical method **Characteristics of analytical method**

SPECIFICITY The chromatogram of the test substance solution showed one test substance peak. The chromatogram of the blank accuracy sample showed no peak at the retention time of the test substance. Therefore the analytical method was found to be specific for the substance.

LINEARITY There was a linear relationship between response and test substance concentration in the range of 79.9 – 1200 mg/L. Since the coefficient of correlation (r) was > 0.99 and the back calculated accuracies were in the range 85-115% the regression line was accepted. Regression line: see attached document. - Slope: 6.80E01 - Intercept: -1.55E02 - Weighting factor: 1/concentration^2 - r: 0.99993

ACCURACY AND REPEATABILITY The mean accuracy at each concentration level fell in the criterion range of 70-110% and the coefficient of variation was = 20% (see

### Endpoint study record: Analytical methods. HPLC-RI. WIL Research Project 500674

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... net Files/Content.Outlook/6RSURWTF/Endpoint study record Analytical methods_ HPLC-RI_ WIL Research Project 500674.html [9/13/2013 2:27:56 PM] table below). The analytical method was therefore accepted for the analysis of the test substance in water in the target concentration range of 100 - 1000 mg/L.

LIMIT OF QUANTIFICATION The LOQ was assessed at 100 mg/L in water.

STABILITY OF THE ANALYTICAL SYSTEM AND END SOLUTIONS - CV (responses) over a 19.5-h time interval at 79.9 mg/L nominal: 1.7% (n=8) - CV (responses) over a 19.5-h time interval at 156 mg/L nominal: 1.5% (n=8) - CV (responses) over a 18.1-h time interval at 1200 mg/L nominal: 1.3% (n=8) Since the CV values were = 20% the analytical system and end solutions were found stable over at least a 18.1-h time interval.

STABILITY OF THE STOCK SOLUTIONS Because the CV (response factors) of the calibration solutions prepared with fresh and stored stock solutions was = 10% (2.1% was measured) the stock solutions were stable when stored at room temperature for at least 1 day.

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. 

## Independent laboratory validation (if applicable) Any other information on results incl. tables

Accuracy and Repeatability

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

Storage stability of samples in the freezer

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



# Overall remarks, attachments Attached background material

Attached document Remarks HPLC chromatograms and Regression line-TS 204240.pdf / 23.61 KB (application/octet-stream) HPLC-RI chromatogram of 156 mg/L calibration solution and of blank accuracy sample + Regression line 

# Applicant's summary and conclusion
Conclusions

### Endpoint study record: Analytical methods. HPLC-RI. WIL Research Project 500674

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... net Files/Content.Outlook/6RSURWTF/Endpoint study record Analytical methods_ HPLC-RI_ WIL Research Project 500674.html [9/13/2013 2:27:56 PM] For the quantitative analysis of 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) in water, an HPLC-RI method was developed and successfully validated for the following parameters:
Specificity: specific Linearity (79.9 – 1200 mg/L, nominal): r = 0.99993 Accuracy (100 and 1000 mg/L, nominal): 106 and 104% Repeatability (100 and 1000 mg/L, nominal): 1.8 and 1.3% Limit of quantification: 100 mg/L Stability analytical system and end solutions (room temp. 18.1 h): stable Stability stock solutions (room temp., 1 day): stable Storage stability (= -15°C, at least 16 h): stable
The study has been performed according to EC recommendations and in compliance with the principles of GLP.