Cpsd-An-00175-Mthd-dimethyl Fumarate 2009 251 Ec French Arrete & Kc Mark-V10

DIMETHYL FUMARATE BY GAS

CHROMATOGRAPHY 2012/412/EC, FRENCH ARRETE & KOREA KC

MARK

CPSD-AN-00175-MTHD

Approval Date: 15 July 2013

Version No.: 10

Business Line: Analytical

PROPRIETARY INFORMATION: This method has been prepared by and is the sole property of Bureau Veritas Consumer Products Services, Inc. ("BVCPS"). The holder may not copy, reproduce, or disclose to a third party without the prior written consent of BVCPS. This method is subject to change without notice.

*Mandatory Field to be filled in. CPSD-AN-00175-MTHD-DIMETHYL FUMARATE 2009 251 EC FRENCH ARRETE & KC MARK-V10 of 11

1. Purpose*:

1.1 This method describes a procedure for the determination of dimethyl fumarate by using Gas Chromatography Mass Spectrometry (GC-MSD)

2. Scope*:

2.1 This method is applicable to all materials except metal, glass and ceramic. However, it is upon request for doing metal, glass and ceramic

2.2 REACH Annex XVII, item 61 (2012/412/EC), French Arrete and Korea KC mark, safety requirement for toxic substances.

2.3 For Korea KC mark, safety requirement for toxic substances, it is only applicable to the part where leather materials are used for children and adult (not for infant ), including inner wears, middle wears, outer garments and bed clothes. “Other products’” are not worn directly and custom-tailored are not subject to the safety requirement for toxic substances

3. Definitions:

3.1. Not applicable

4. Apparatus and Equipment:

4.1 Analytical balance capable of weighing to 0.001g or better resolution

4.2 Ultrasonic bath, with temperature control

4.3 Gas Chromatography / Mass Spectrum Detector, Agilent HP6890 & MS5973 or equivalent performance

4.4 Zebron ZB-35 Capillary GC, 15m x 0.25mm ID, 0.5µm or equivalent performance

4.5 DB-35MS, 30 x 0.25mm x 0.25 µm, J&W or equivalent performance

4.6 General laboratory apparatus

5. Reagents, and Consumables:

All chemicals and reagents shall be in analytical grade unless otherwise specified.

5.1 Standards

5.1.1 Dimethyl fumarate CAS No.: 624-49-7 (ChemService or equivalent quality)

5.1.2 Dimethyl maleate CAS No.: 624-48-6 (Sigma or equivalent quality)

5.2 Internal standard

5.2.1 Naphthalene-d8 – Internal standard

5.3 Reagent and solvent

5.3.1 Ethyl Acetate

Noted: Check the solvent background in GCMS before use, different Brand name may have different solvent background performance, and huge solvent background have possibility masking analyst peak with low concentration

5.4 Standard solutions

5.4.1 Individual stock standard solution (1000 µg/mL)

CPSD-AN-00175-MTHD

.*Mandatory Field to be filled in. CPSD-AN-00175-MTHD-DIMETHYL FUMARATE 2009 251 EC FRENCH ARRETE & KC MARK-V10 of 11

Weigh accurately 0.01g (10mg) of dimethyl fumarate (5.1.1) and dimethyl maleate (5.1.2) into each individual beaker, dissolve in a portion of ethyl acetate (5.3.1) and then transfer into a 10mL volumetric flask individually and make up to the mark with ethyl acetate (5.3.1)

5.4.2 Individual intermediate standard solution (10 µg/mL)

Pipette 0.1mL of 1000µg/mL individual stock standard solution (5.4.1) into each individual 10mL volumetric flask and make up to the mark with ethyl acetate (5.3.1)

5.4.3 Individual working standard solution (1 µg/mL)

Pipette 1mL of 10µg/mL individual intermediate standard solution (5.4.2) into each individual 10mL volumetric flask and make up to the mark with ethyl acetate (5.3.1)

5.4.4 Dimethyl Maleate check standard solution (20 ng/mL)

Pipette 0.2mL of 1µg/mL dimethyl maleate working standard solution 1(5.4.3) into a 10mL volumetric flask and make up to the mark with ethyl acetate (5.3.1)

5.5 Internal standard solutions

5.5.1 Internal standard stock solution (1000 µg/mL)

Weigh accurately 0.01g (10mg) of Naphthalene – d8 (5.2.1) into a beaker, dissolve in a portion of ethyl acetate (5.3.1) and then transfer into a 10mL volumetric flask and make up to the mark with ethyl acetate (5.3.1)

5.5.2 Internal standard intermediate solution (100 µg/mL)

Pipette 1mL of Naphthalene – d8 stock internal standard solution, 1000 µg/mL (5.5.1) into a 10mL volumetric flask and make up to the mark with ethyl acetate (5.3.1)

5.5.3 Internal standard working solution for sample dilution (1 µg/mL)

Pipette 1mL of Naphthalene – d8 intermediate internal standard solution, 100 µg/mL (5.5.2) into a 100mL volumetric flask and make up to the mark with ethyl acetate (5.3.1)

5.6 Calibration standard solutions

5.6.1 Working standard solution of dimethyl fumarate

Prepare 3 calibration standard solutions (5, 20 and 100ng/mL with 1mg/L internal standard respectively) according to the following table:

Std Volume of working standard solution of

dimethyl fumarate (5.4.3) (1µg/mL), mL

Volume of internal standard intermediate solution (5.5.2) (100

µg/mL), mL

Make up volume (ethyl acetate),

mL

Concentration, ng/mL (ppb)

1 1 0.1 10 100

2 0.2 0.1 10 20

3 0.05 0.1 10 5

6. Quality Control:

6.1 For Quality control, refer to CPSD-AN-TSOP-QUALITY CONTROL FOR TESTING

6.2 For standard solution storage life time, refer to CPSD-AN-TSOP-PREPARATION OF STANDARD SOLUTIONS

7. Interferences:

7.1 Not applicable

8. Safety and PPE Information:

8.1 P:\CPSDOC\Health Safety Environment\FORM\CPSD-GB-HSE-FORM-RISK ASSESSMENT AND REQUIRED PPE TABLE

CPSD-AN-00175-MTHD


8.2 For General Chemical Laboratory Safety Requirements, refer to corresponding section of “CPSD Hazard Method” (GB MTHD)

8.3 Hazard table

Hazard Sub Hazard(s) Injury(ies) Preventive Measure(s) PPE(s) CPSD Hazard Method

Dangerous Good

GC with various detectors (i.e. ECD, MS and etc)

Skin, eye, and respiratory irritation.

1. Regularly observe the operation of instrument to ensure it functions properly. 2. Ventilation and exhaust MUST be sufficient during instrument operation. 3. In case there is abnormal found in electrical equipment, stop the operation and switch off the electricity supply if it is safe to do so. Report to supervisor and call engineer for repair. 4. Open the door for minutes before entering the instrumental area if ventilation and exhaust are suspected out of service. Stop the analysis and report to supervisor for repair.

General lab safety equipment: Safety glasses, chemical resistant boots, gloves and etc.

CPSD-GB-00037-MTHD-DANGEROUS GOODS HAZARD

Organic solvent (i.e. Methanol, acetone, acetonitrle, tetrahydrofuran, ethanol, hexane, pentane, benzene, toluene and etc)

Handle with care in fumehood

9. Method:

9.1 Sample preparation

9.1.1 Cut the sample into small pieces, approximately 5mm x 5mm

9.1.2 Weigh to nearest 1 mg, approximately 1 g of sample into a 40 ml reaction vial with screw cap

9.1.3 Pipette 5mL ethyl acetate (5.3.1) and 0.05mL 100µg/mL internal standard intermediate solution (5.5.2) into vial which containing the sample

(Add sufficient amount of solvent into the vial, if 5mL ethyl acetate cannot immerse the sample)

9.1.4 Cap and place the vial into an ultrasonic bath at 40°C and sonicate for 30minutes

9.1.5 Cool the sample solution to room temperature

9.1.6 Filter the extract by PTFE syringe filter with pore size 0.45µm into a 2mL vial for GCMSD analysis

(If extraction volume more than 5mL, squeeze the sample extract into evaporating tube and concentrate the extract under inert gas to ~2-3mL, then make-up the volume to 5mL with ethyl acetate)

9.2 Preparation of Method Blank

9.2.1 Pipette 5mL ethyl acetate (5.3.1) and 0.05mL 100µg/mL internal standard intermediate solution (5.5.2) into a 40mL vial

9.2.2 Follow sample extraction procedure 9.1.4 to 9.1.6

9.3 Preparation of Sample spike/ Blank spike (0.1ug)

CPSD-AN-00175-MTHD


9.3.1 For spike blank, spike 0.1mL of 1µg/mL dimethyl fumarate working standard solution (5.4.3) into a 40 mL reaction vial. Process step 9.1 without sample involved

9.3.2 For spike sample, spike 0.1mL 1µg/mL dimethyl fumarate working standard solution (5.4.3) into a 40 mL reaction vial with 1 g sample. Process step 9.1

Theoretical Concentration of analytes in spike blank and spike sample

Chemicals Concentration in extract,

µg/mL Concentration in sample,

mg/kg

Dimethyl Fumarate 0.02 0.1

9.4 Instrument analysis

9.4.1 GC-MS Program (The below instrument conditions are reference only and should be used as a guide)

GC: Agilent 6890

Injection Volume: 1 - 2µL (depends on instrument model and sensitivity)

Plunger speed: Fast

Inlet Temp.: 250oC

Injection Mode: Splitless

Liner: Split / Splitless liner (ID: 4mm, single tap, with glasswool)

Column: ZB-35, (15 m x 0.25 mm x 0.5 µm) or DB-35MS (30m x 0.25mm x 0.25 µm)

Carrier Gas: Helium

Carrier gas flow: 1.0 mL/min, (Constant flow)

Oven Temp.: Initial: 50°C for 1 min, Ramp 1: 25°C/min to 125°C for 0 min, Ramp 2: 30°C/min to 300°C for 5 min

Post temperature: 300oC (for DB-35MS only)

Post time: 2 min (for DB-35MS only)

Total Run Time: 14.833 min (for ZB-35) 16.833 min (for DB-35MS)

MSD Transfer Line Heater:

MSD Heater: 300oC

MS: MS 5975

Tune File atune.u

Solvent delay: 3.00 min

EM offset: 400V

MS Quad: 150oC (Max: 200oC)

MS Source: 230oC (Max: 250oC)

Acquisition mode: SCAN / SIM mode

Scan Range: 50 amu – 150 amu

Sampling Rate: 2

Threshold: 150 or 100 (depends on sensitivity)

SIM Acquisition:

Group 1

Resolution: Low

Ion (Dwell) : 59 (50) 85 (50) 108 (50) 113 (50)

136 (50) 137 (50)

CPSD-AN-00175-MTHD


Characteristics for GC-MS analysis

Substances

RT

(min) Target ion

(m/z)

Reference ions

(m/z) ZB-35 DB-35

Naphthalene- d8 (Internal standard)

~5.3 ~6.26 136 108, 137

Dimethyl Fumarate ~4.19 ~5.03 113 59, 85

Dimethyl Maleate ~4.43 ~5.25 113 59, 85 Remark: retention time of each isomer are reference only and may have time shift after column cutting or connected with vent free / backflush

9.4.2 Calibration

9.4.2.1 Calibrate the GC-MSD with calibration standard solutions prepared in 5.6.1

9.4.2.2 Inject one point of dimethyl maleate (20ng/mL) prepared in 5.4.4

Remark: To locate the retention time of dimethyl maleate isomer and evaluate column resolution, not for quantitation

9.4.3 Sample Measurement

9.4.3.1 Analyze the method blank and the sample extract. From the calibration curve, read the concentration of dimethyl fumarate in sample solution.

9.4.3.2 Dilute the sample with 1µg/mL - Internal standard working solution (5.5.3) if the concentration is out of the calibration range

9.4.4 Calculation

Analyte in sample (mg/kg) = Conc. read from instrument (ng/mL) x Final vol. (mL) x Dilution factor

Sample weight (g) x 1000

10. Reporting Criteria*:

10.1 Requirement

According to French Arrete,

Parameter Maximum Allowable Limit

Dimethyl Fumarate Not Detected

According to

(i) REACH Annex XVII, item 61 (2012/412/EC) and

(ii) Korea KC mark, safety requirement for toxic substances,

Parameter Maximum Allowable Limit

Dimethyl Fumarate 0.1mg/kg

10.2 The laboratory reporting limit

Parameter Laboratory Reporting Limit

Dimethyl Fumarate 0.03 mg/kg

10.3 The Method detection limit: 0.03 mg/kg

10.4 Data sheet

10.4.1 The data sheet shall include the followings:

a) Unique reference no., e.g., application no., sample no., report no., etc.

CPSD-AN-00175-MTHD


b) Standard/ procedure performed

c) Sample ID

d) Sample description

e) Sample weight / surface area

f) GC-MSD raw data

g) Analytes content in sample

h) Identity of equipment used including: balance, GC-MSD, etc.

i) Identity of personnel performing the specific task and date:

i. Sample preparation

ii. Sample extraction

iii. Calculation

iv. Result checking

11. Measurement Uncertainty:

11.1 Measurement uncertainty needs to be calculated by location themselves if necessary

12. Literature References:

12.1 Korea Safety Quality Mark – Annex 1: Textile Products

12.2 Korea Safety Standards for Self-Regulatory safety confirmation products – Annex 4B: Textile Products for Infant – Dimethyl fumarate quantitative method

12.3 Commission Regulaiton (EU) No. 412/2012 of 15 May 2012 amending Annex XVII to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

13. Modifications from Standard:

13.1 For Korea KC mark, modified the extraction solvent & extraction method and use of internal standard

14. Associated Documents:

14.1. CPSD-AN-TSOP-QUALITY CONTROL FOR TESTING

14.2. CPSD-AN-TSOP-PREPARATION OF STANDARD SOLUTIONS

14.3. CPSD-AN-00429-MTHD-KOREA CERTIFICATION MARK

14.4. CPSD-AN-00680-MTHD-METHOD SUMMARY OF REQUIREMENTS FOR BAGS CASES & TRUNKS CNS 15331

14.5. CPSD-GB-00037-MTHD-DANGEROUS GOODS HAZARD

15. Annex:

Appendix 1 – Chromatogram and spectrum of Dimethyl Fumarate and Dimethyl Maleate with ZB-35 column

CPSD-AN-00175-MTHD


CPSD-AN-00175-MTHD


Appendix 2 – Chromatogram and spectrum of Dimethyl Fumarate and Dimethyl Maleate with DB-35MS column

CPSD-AN-00175-MTHD


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CPSD-AN-00175-MTHD


Name Date

Author: Mantak Ho 05 July 2013

Approver: Van Mak 15 July 2013

Approver: Joerg Ruhkamp 12 April 2013

CPSD-AN-00175-MTHD


REVISION HISTORY Creation Date of Version 1: Januar y 15, 2009

Ver. No. Section Changes Approval Date

2

-

9

10

Amended the Title

Amended the typo

Amended the extraction method for absorbent samples

Updated the requirement and reporting limit

April 8, 2009

3

All

2

Amened the typo

Amended the scope May 11, 2009

4

4 Amended the column for separation of dimethyl fumarate and dimethyl maleate

November 6, 2009

5 Amended the column for separation of dimethyl fumarate and dimethyl maleate 6 Added the standard of dimethyl maleate 9 Amended the calibration range

9 Amended the preparation of standard and interpretation of dimethyl fuamrate and dimethyl maleate

10 Added the method detection limit 14 Added chromatograms

5 6, 8, 11, 14, 15

1. New CPSD template revise (Added with PPE information and Annex)

2. Quality Control revise (refer to CPSD-AN-TSOP-QUALITY CONTROL FOR TESTING for general)

3. remove batchwise duplicate practice

27 April 2010

6

2 Added the scope for KC mark

30 July 2010 10 Added the requirement of KC mark 12 Added the literature references of KC mark 13 Added the details for modifications of KC mark Standard 14 Added the associated documents for KC mark

7

4, 5

6, 9

14, 15

1. Added new GCMS program setup for DB-35MS column

2. Added Chromatogram and spectrum with DB-35MS column for reference

3. Minor amendment

1 February 2012

8 2, 4, 5, 10, 12

Title and filename: Updated citation from 2009/251/EC to REACH Annex XVII, item 61 (2012/412/EC) 29 May 2012

9 8, 14 Added safety and PPE information and associated documents

Mantak Ho, 11 April 2013 12 April 2013

10 8, 14 Added safety and PPE infromation and associated documents

Mantak Ho, 05 July 2013 15 July 2013

CPSD-GP-MTD-RREF-MASTER METHODS TEMPLATE-V9 DOI: July 4, 2013

Cpsd-An-00175-Mthd-dimethyl Fumarate 2009 251 Ec French Arrete & Kc Mark-V10

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