1. Bonnie Tay-Jones Yen Ping Principal Research Officer Quality and Environment Assessment Unit Malaysian Palm Oil Board 6 th International Conference and Exhibition on Analytical & Bioanalytical Techniques Gas Chromatography-flame ionization detection of 1,4-dioxane in palm oil-based fatty alcohol ethoxylates

2. M ALAYSIAN P ALM O IL B OARD

3. Malaysian Palm Oil Board Vision To become the premier, research and development institution providing leadership and impetus for the development of a highly diversified, value-added, globally competitive and sustainable oil palm industry. Mission To enhance the well-being of the Malaysian oil palm industry through research, development and excellent services.

4. A BBREVIATIONS GC-FID – Gas Chromatography-flame ionization detector FAEO – fatty alcohol ethoxylate I & I – industrial and institutional EO – ethylene oxide S/N – signal-to-noise ratio LOD- limit of detection LOQ – limit of quantification RSD – relative standard deviation AOAC – Association of Official Analytical Chemists FDA – Food & Drug Administration (USA) NIST – National Institute of Standards and Technology

5. Introduction 1,4-dioxane the issue 1,4-dioxane origin Fatty alcohol ethoxylate and its applications 1,4-dioxane toxicity Regulatory Limit Methodology Sample preparation Instrumentation Validation of method Validation Parameters LOD, LOQ, Precision, Accuracy, Specificity Confirmation of 1,4- Dioxane Identification of 1,4-dioxane in fatty alcohol ethoxylate by GC-MSD OVERVIEW

6. INTRODUCTION

7. Production of oleochemical derivatives :current developing industry in Malaysia Local oleochemical companies: setting up of new commercial plant to produce palm-based fatty alcohol ethoxylates (FAEO). Research group in AOTD,MPOB currently setting a pilot plant producing ethoxylates. 1,4-dioxane presence : levels up to 279 ppm*** in personal care products formulated with ethoxylated products e.g alcohol ethoxylate, alcohol ethoxy sulfates, polyethylene glycol and polysorbates (Black, 2001***). FDA,USA (2001) : detected up to 1410 ppm in cosmetic raw materials 1,4- DIOXANE : T HE ISSUE ***Black RE, Hurley FJ, Havery DC. 2001. Occurrence of 1,4-dioxane in cosmetic raw materials and finished cosmetic products. Journal of AOAC International 84(3): 666-670.

8. 1,4- DIOXANE : THE ISSUE 1,4-dioxane : recognized as a toxic substance, need to be controlled. ( realised by local manufacturer) A request for method to be developed by Quality and Environment Unit, Advanced Oleochemical Technology Division Method for detection of 1,4-dioxane in palm- based FAEO not available. Local industry and MPOB research unit need to perform quality control of products for customer requirements.

9. 1,4- DIOXANE ORIGIN A by-product that can be produced during the manufacturing of fatty alcohol ethoxylates. Ethoxylation Process: Addition of ethylene oxide to a linear alkyl fatty alcohol (carbon chains,C12-C14) normally derived from palm kernel oil/coconut oil. 1,4-dioxane may be formed due to dimerization of ethylene oxide (under certain reaction conditions). Continue…

10. C HEMICAL S TRUCTURES 1,4-dioxane Fatty alcohol ethoxylate n = number of moles EO

11. C OMMERCIAL PALM - BASED ALCOHOL ETHOXLYATES WITH DIFFERENT EO COMPOSITIONS FAEO 20 FAEO 12 FAEO 2 FAEO 1 FAEO 4FAEO 3

12. F ATTY ALCOHOL ETHOXYLATE APPLICATIONS

13. F ATTY ALCOHOL (C12-C14) ETHOXYLATES WITH 1 – 6 MOLES ETHYLENE OXIDE APPLICATIONS Source: Thai Ethoxylate Co. Ltd, Thailand MolePersonal careHome Care Paint and rubber AgrochemicalsTextile Metal working shampoo Shower cream/gel Hand cleaner Laundry dtergent (powder) Laundry detergent (liquid) Hard surface cleaners Dishwash detergent Household cleaner I & I cleaners Emulsion polymerization Herbicide/fungicide inseciticide fertilizer Scouring agent Wetting agent Degreasing agent 1 2 3 4 5 6

14. MolesPersonal careHome Care Paint and rubber AgrochemicalsTextile Metal working shampoo Shower cream/gel Hand cleaner Laundry detergent (powder) Laundry detergent (liquid) Hard surface (cleaners) Dishwash detergent Household cleaner I & I cleaners Emulsion polymerization Herbicide/fungicide inseciticide fertilizer Scouring agent Wetting agent Degreasing agent 7 8 9 10 11 12 15 Fatty alcohol (C12-C14) ethoxylates with 1 – 6 moles ethylene oxide applications

15. T OXICITY OF 1,4- DIOXANE International Agency for Research on Cancer (IARC) Section 5, pg.595, Vol.71, 1999 5.1 Exposure data Exposure to 1,4-dioxane may occur during its manufacture and its use as a solvent in a wide range of organic products. It has been detected in ambient air. 5.2 Human carcinogenicity data Deaths from cancer were not elevated in a single, small prospective study of workers exposed to low concentrations of dioxane. 5.3 Animal carcinogenicity data Oral administration and inhalation exposure study in mice, rats and guinea-pigs: increased incidence of tumour occurrence in the tested animals. Continue…..

16. 5.5 Evaluation There is inadequate evidence in humans for the carcinogenicity of 1,4-dioxane. There is sufficient evidence in experimental animals for the carcinogenicity of 1,4-dioxane. Overall evaluation 1,4-Dioxane is possibly carcinogenic to humans (Group 2B). Continue…

17. R EGULATORY L IMIT No regulatory limit for 1,4-dioxane in palm-based fatty alcohol ethoxylate produced in Malaysia International cooperation on cosmetics regulation (ICCR), an international group of regulatory authorities for cosmetics (Japan, United States, Canada and European Union) : proposal to set exposure level limits in cosmetic and personal care products. 8 th ICCR meeting held on the July, 2014: the reports on 1,4-dioxane in cosmetic products are undergoing final review

18. M ETHOD D EVELOPMENT

19. M ETHOD AND PERFORMANCE EVALUATION Method Name: Determination of 1,4-Dioxane in fatty alcohol ethoxylates by using GC-FID Method performance: assessed by doing a method validation based on International Committee on Harmonization (ICH) /AOAC guidelines Matrices for spiking/recovery: palm-based fatty alcohol ethoxylates with different EO composition

20. S AMPLE PREPARATION FOR A NALYSES (D IRECT INJECTION ) 0.5 g of FAEO sample weigh into volumetric flask (5 ml) Inject into GC-FID reconstitute with acetonitrile Note: Fast sample preparation and no clean up of matrices required (cost saving method)

21. A GILENT T ECHNOLOGIES 7890 GC-F LAME IONISATION DETECTOR

22. GC-FID CONDITIONS FOR DETECTION OF 1,4-D IOXANE GC oven temperature program : initial temperature was (50 o C) held for 4 min, then increased at 10 o C/ min to 110 o C (held 20 min). Post Run, 300 o C held for 10 min. Total run time was 34 minutes. Carrier gas: helium (set at constant flow of 0.8 mL/ min) Inlet temp, Pressure: 200 o C; 4.47 psi Detector temp : 310 o C Split ratio: 10:1 Column: HP-5 column (30 m length x 0.32 mm internal diameter (i.d.); Agilent Technologies)

23. GC-FID SAMPLING CONDITIONS OPTIMIZATION FOR DIRECT INJECTION A split focus liner with deactivated glass wool was used at the inlet port, and replaced if contaminated over time. The glass wool in the liner is able to trap the heavier ethoxylates and only allow the volatiles through (prevent matrix interference). Additional post run for 10 mins at 300 o C was included after every analysis to remove other volatile residues (arising from solvent and matrixs) from the column.

24. I NSTRUMENTATION – STRUCTURE CONFIRMATION GC conditions from GC-FID method was transferred to an Agilent Technologies 7890A GC fitted with 5975 C Mass spectrometric detector Data handling and system operations controlled by GC-MS NISTO5 software. Mass spectra obtained were obtained for 1,4- dioxane spiked in FAEO.

25. M ETHOD V ALIDATION Limit of detection Limit of quantification Precision Linearity and working range Accuracy Selectivity

26. 1 ug/ml 1,4-dioxane RT : 8.589 mins FAEO (blank) FAEO ( spiked at 1 ug/ml) RT 1,4-dioxane : 8.583 mins GC-FID chromatograms of 1,4-dioxane, blank FAEO & spiked FAEO

27. L OD / LOQ LOD & LOQ : based on S/N ratio approach Acceptable S/N ratio for LOD was 3:1 and 10:1 for LOQ (ICH guideline) For this method: LOD : 10 µg/g of FAEO LOQ : 30 µg/g of FAEO

28. * Same analyst, same instrument Evaluation Criterion: AOAC Guidelines at ppm level, acceptable RSD for ppm : < 6% P RECISION ( INTRA DAY ) Concentration of 1,4-dioxane (μg/g)* n= 4 Percentage Recovery (%) RSD (%) 15.5 (low)99.40.4 77.5 (medium)100.60.4 505.0(high)101.40.2 Table 2 Intra-day precision results and statistical data

29. INTERMEDIATE PRECISION 2 conditions: same analyst on different days; different analyst on different days n= 6 replicates Evaluation Criterion: AOAC Guidelines at ppm level, acceptable RSD for ppm : < 6% Inter-day and intermediate precision for 1,4-dioxane at 100 µg/g n = 6 RSD (%) Day 10.5 Day 20.4 Analyst 10.5 Analyst 20.4

30. L INEARITY AND WORKING RANGE A series of 6 calibration solution were prepared at the LOD up to the highest expected working concentration. These standards were run for 6 different batches. Linear Regression Data 1,4-dioxane working range (5.0 – 700 µg/g) n = 6 Slope0.6524 Intercept0rigin Standard error0.05 R 2 value0.9999

31. A CCURACY o Accuracy estimated by using recovery studies (spiking 1,4-dioxane in FAEO) o Evaluation criterion for accuracy : 80 – 115% (AOAC Guidelines) 1,4-dioxane (μg/g) FAEO (3 moles EO) FAEO (7 moles EO) FAEO (9 moles EO) Recovery (%) N= 6 RSD (%) Recovery (%) N = 6 RSD (%) Recovery (%) N =6 RSD (%) 30 103.51.5 99.92.4 96.82.3 60 101.42.5 100.02.4 98.42.9 100 99.91.3 98.01.5 96.93.3 200 101.82.2 97.32.9 96.21.9 500 104.33.0 96.53.8 97.22.1

32. M ONITORING OF COMMERCIAL FAEO WITH DIFFERENT ETHYLENE OXIDE COMPOSITIONS 22 types of commercial FAEO samples from local and overseas manufacturing companies with various ethylene oxide compositions (1,2, 3, 5,7, 9,12 and 20) were found to be free from 1,4-dioxane.

33. M ASS SPECTRUM OF SPIKED FATTY ALCOHOL ETHOXYLATE OBTAINED FROM 7890 A GILENT T ECHNOLOGIES GC-5975C T RIPLE A XIS MSD Spectrum matching from experiment (spiked at 500 µg/g ) and NIST library GC-MSD: 94% Mw: 88.1 (for 1,4-dioxane)

34. C ONCLUSIONS Method is fit for the detection of 1,4-dioxane in palm- based fatty alcohol ethoxylate with different moles ethylene oxide at LOD of 10 µg/g of FAEO within the range of 5 µg/g to 700 µg/g with precision < 6%, and accuracy within 80 - 115% Method will be useful for routine monitoring of 1,4- Dioxane,due to its simple preparation,and the use of a commonly available instrument (GC-FID) at the Quality control laboratory of local FAEOs producers. Local and overseas commercial FAEOs were found to be free from 1,4-dioxane.

35. F URTHER INFORMATION Gas Chromatography with flame ionization detection of 1,4-dioxane in palm-based fatty alcohol ethoyxlates Bonnie Tay Yen Ping ; Zulina A Maurad and Halimah Muhammad JAOCS, 2014, 91, 1103 – 1110.

36. Committee Members of 6th Bioanalytical conference –for their kind invitation to present at this conference Director General of Malaysian Palm Oil Board for permission to present this work Malaysian Palm Oil Board – for funding this research Thai Ethoxylate Ltd, Bangkok, Thailand – providing fatty alcohol ethoxylates samples for spiking/recovery work A CKNOWLEDGEMENT

37. Thank you