Bonnie Tay-Jones Yen Ping

Principal Research Officer

Quality and Environment Assessment Unit

Malaysian Palm Oil Board

6th International Conference and Exhibition on Analytical & Bioanalytical Techniques

Gas Chromatography-flame ionization detection of 1,4-

dioxane in palm oil-based fatty alcohol ethoxylates

MALAYSIAN PALM OIL BOARD

Malaysian Palm Oil Board

VisionTo 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.

MissionTo enhance the well-being of the Malaysian oil palm industry through research, development and excellent services.

ABBREVIATIONS 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

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

INTRODUCTION

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 : THE 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.

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.

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…

CHEMICAL STRUCTURES

1,4-dioxane Fatty alcohol ethoxylaten = number of moles EO

COMMERCIAL PALM-BASED ALCOHOL ETHOXLYATES WITH DIFFERENT EO COMPOSITIONS

FAEO 20 FAEO 12

FAEO 2

FAEO 1FAEO 4FAEO 3

FATTY ALCOHOL ETHOXYLATE APPLICATIONS

FATTY ALCOHOL (C12-C14) ETHOXYLATES WITH 1 – 6 MOLES ETHYLENE OXIDE APPLICATIONS

Source: Thai Ethoxylate Co. Ltd, Thailand

Mole Personal care Home Care Paint and rubber

Agrochemicals Textile 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

Moles Personal care Home Care Paint and rubber

Agrochemicals Textile 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

TOXICITY OF 1,4-DIOXANEInternational 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 dataOral administration and inhalation exposure study in mice, rats and guinea-pigs: increased incidence of tumour occurrence in the tested animals.

Continue…..

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…

REGULATORY LIMIT

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.

•8th ICCR meeting held on the July, 2014: the reports on 1,4-dioxane in cosmetic products are undergoing final review

METHOD DEVELOPMENT

METHOD 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

SAMPLE PREPARATION FOR ANALYSES (DIRECT 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)

AGILENT TECHNOLOGIES 7890 GC-FLAME IONISATION DETECTOR

GC-FID CONDITIONS FOR DETECTION OF 1,4-DIOXANE

GC oven temperature program :initial temperature was (50oC) held for 4 min, then increased at 10oC/ min to 110oC (held 20 min). Post Run , 300oC 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: 200oC; 4.47 psi Detector temp : 310oC Split ratio: 10:1 Column: HP-5 column (30 m length x 0.32 mm

internal diameter (i.d.); Agilent Technologies)

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 300oC was included after every analysis to remove other volatile residues (arising from solvent and matrixs) from the column.

INSTRUMENTATION – 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.

METHOD VALIDATION

Limit of detection Limit of quantification Precision Linearity and working range Accuracy Selectivity

1 ug/ml 1,4-dioxaneRT : 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

LOD /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 FAEOLOQ : 30 µg/g of FAEO

* Same analyst, same instrument

Evaluation Criterion: AOAC Guidelines at ppm level, acceptable RSD for ppm : < 6%

PRECISION (INTRA DAY)

Concentration of 1,4-dioxane

(μg/g)*n= 4

Percentage Recovery (%)

RSD (%)

15.5 (low) 99.4 0.4

77.5 (medium) 100.6 0.4

505.0(high) 101.4 0.2

Table 2 Intra-day precision results and statistical data

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 1 0.5

Day 2 0.4

Analyst 1 0.5

Analyst 2 0.4

LINEARITY 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

Slope 0.6524

Intercept 0rigin

Standard error 0.05

R2 value 0.9999

ACCURACYo 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)

 

  Recover

y

(%)

N= 6

RSD

(%)

  Recovery

(%)

N = 6

RSD

(%)

Recovery

(%)

N =6

RSD

(%)

30 103.5 1.5   99.9 2.4   96.8 2.3

60 101.4 2.5   100.0 2.4   98.4 2.9

100 99.9 1.3   98.0 1.5   96.9 3.3

200 101.8 2.2   97.3 2.9   96.2 1.9

500 104.3 3.0   96.5 3.8   97.2 2.1

MONITORING OF COMMERCIAL FAEO WITH DIFFERENT ETHYLENE OXIDECOMPOSITIONS

• 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.

MASS SPECTRUM OF SPIKED FATTY ALCOHOL ETHOXYLATE OBTAINED FROM 7890 AGILENT TECHNOLOGIES GC-5975C TRIPLE AXIS MSD

Spectrum matching from experiment (spiked at 500 µg/g ) and NIST library GC-MSD: 94%

Mw: 88.1 (for 1,4-dioxane)

CONCLUSIONS

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.

FURTHER INFORMATION

Gas Chromatography with flame ionization detection of 1,4-dioxane in palm-based fatty alcohol ethoyxlatesBonnie Tay Yen Ping ; Zulina A Maurad and Halimah Muhammad

JAOCS, 2014, 91, 1103 – 1110.

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

ACKNOWLEDGEMENT

Terima kasih

Thank you