### Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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Prepared Checked/Approved valid as of

H. Meuth DI M. Hirschen 08.05.09 Title: 

### Quantitative determination of Decanoyl oxy benzoic acid

(DOBA) and Hydroxy benzoic acid (HBA) in DOBA sampl es by HPLC

Contents
1. Objective and scope 2. Principle 3. Apparatus 4. Reagents 5. Procedure 6. Validation of the method 7. Remarks 8. Further applicable documents 9. Literature references 10. Appendix

Validation: Precision

Accuracy

________________________

________________________

### Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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1. Objective and scope
The method is used to determine the content of deca noyl oxy benzoic acid (DOBA) and hydroxy benzoic acid (HBA) in DOBA samples by HPLC.

2. Principle The DOBA and HBA content are determined by reversed phase HPLC with UV detection.

3. Apparatus HPLC instrument with dual gradient pump, e. g. Agil ent 1100 UV detector, e. g. Agilent 1100 HPLC column, Synergi Polar RP, 250 mm x 4,6 mm ID, 4 μm (Phenomenex, No. OOG-4336-EO)
Ultrasonic bath Ordinary laboratory equipment

4. Reagents 4.1 Acetonitrile, HPLC grade, e.g. Riedel de Haen N o. 34851 4.2 2-Propanol, HPLC grade, e. g. Riedel de Haen No . 34863 4.3 Tetrabutyl ammonium hydrogen sulphate (TBAHS), puriss., e. g. Fluka No. 86868
4.4 Solvent 1: 2-propanol / acetonitrile / water = 5 / 3 / 2 (v/v/ v)
4.5 Solvent 2: acetonitrile / water = 1 / 1 (v/v)
4.6 Eluent A: water / acetonitrile = 95 / 5 (v/v) + 0,005 mol/l T BAHS
4.7 Eluent B: water / acetonitrile = 5 / 95 (v/v)
4.8 DOBA reference substance with known DOBA conten t, e. g. BES 04101, w = 99,9 % (w/w) 4.9 4-Hydroxybenzoic acid reference substance with known content, e. g. Aldrich No. 240141

### Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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5. Procedure 5.1 Sample preparation Weigh to the nearest 0,1 mg an appropriate amount o f the test substance (e. g. approx. 50 mg dry filter cake and approx. 60 mg wet filter cake, resp ectively) into a 10 ml measuring flask. Add approx. 8 ml solvent 1 (4.4) and treat with ult rasound until the substance is completely dissolved. Let the solution cool to room temperature and fill to the mark with solvent 1 (4.4) Dilute sample solution 1:10 with solvent 2 (4.5). Inject 10 μl of the final sample solution.

5.2 Calibration
5.2.1 DOBA Weigh to the nearest 0,1 mg approx. 20 mg, 40 mg an d 60 mg DOBA reference substance (4.8) each into a 10 ml measuring flask. Add approx. 8 ml solvent 1 (4.4) and treat with ult rasound until the substance is completely dissolved. Let the solutions cool to room temperature and fill to the mark with solvent 1 (4.4) Dilute each stock solution 1:10 with solvent 2 (4.5 ). Inject 10 μl of each final calibration solution.

5.2.2 HBA Weigh to the nearest 0,1 mg approx. 35 mg and 65 mg HBA reference substance (4.9) each into a 100 ml measuring flask. Dissolve and fill to the mark with solvent 1 (4.4). Dilute each stock solution 1:1000, 1:5000 and 1:100 00 with solvent 2 (4.5). Inject 10 μl of each final calibration solution.

### Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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5.3 Evaluation Calculate all final solutions in mg/l. Evaluation i s done by external standard calibration via linear regression line.

m

100

a b A w i i ⋅ − =

Where is:

w i content of DOBA and HBA, respectively in % (w/w) A i peak area of DOBA and HBA, respecitvely a slope of regression line b axis intercept of regression line m sample weight in final sample solution in mg/l

5.4 HPLC-Conditions Chromatography column/s: Stationary phase length [mm] ID

[mm] particle

size [μm] Pre-column: none --- --- --- Column: Synergi Polar RP 250 4,6 4

Mobile Phase:
A: water / acetonitrile = 95 / 5 (v/v) + 0,005 mol/l T BAHS (4.6) B: water / acetonitrile = 5 / 95 (v/v) (4.7)
C: ---

Gradient: T [min] % B isocratic: A: --- %
0 30 B: --- %
5 30 C: --- %
15 90
20 90
22 30
30 30

Run time: 30 min Flow rate: 1,0 ml/min

### Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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Column temperature: 23 °C Injection volume: 10 μl

Detection:
UV detector Response time: >0,05 s
DA detector Slit: 4 nm Wavelength/BW: 254 / 4 nm Reference wavelength/BW: 400 / 100 nm

6. Validation of the method
Precision 6-fold determination of DOBA content in sample CUS 06-597:

n 1 : 99,2 n 4 : 99,3

n 2 : 99,1 n 5 : 97,7

n 3 : 99,7 n 6 : 99,9 mean: 99,2 std. deviation: 0,77 Coeff. of variation: 0,8

Precision 6-fold determination of HBA content in sample CUS 0 6-597:

n 1 : 0,07 n 4 : 0,06

n 2 : 0,07 n 5 : 0,06

n 3 : 0,07 n 6 : 0,07 mean: 0,07 std. deviation: 0,005 Coeff. of variation: 7,7

### Clariant Produkte (D) GmbH Laboratory Method LM-D562-CH-0004

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Linearity of DOBA calibration: Conc. [mg/l] area [u] Slope: 6,3936 u/(mg/l) 202,797 1393,35541 Intercept: 102,9990 u 415,584 2772,73975 Correlation coeff.: 0,9999 622,377 4075,79541 Residual std. deviation: 15,5162 u Method std. deviation: 2,4268 u/(mg/l) Rel. method std. dev.: 0,59 %

Linearity of HBA calibration: Conc. [mg/l] area [u] Slope: 59,3891 u/(mg/l) 0,071 7,20713 Intercept: 2,8871 u 0,122 10,17547 Correlation coeff.: 0,9998 0,357 23,76888 Residual std. deviation: 0,2486 u 0,608 39,17189 Method std. deviation: 0,0042 u/(mg/ l) Rel. method std. dev.: 1,45 %

7. Remarks For the determination of by-products, e. g. C 8 - and C 9 -OBA a DOBA calibration is used since no reference materials exist. For the calibration of t he by-products determination 20 mg, 40 mg and 60 mg DOBA reference substance are each dissolved i n 100 ml solvent 1 (4.4) and diluted 1/10, 1/100 and 1/1000 with solvent 2 (4.5) in the same w ay as described for the DOBA calibration.

8. Further applicable documents

none

9. Literature references

none

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10. Appendix Typical Chromatogram: