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Natural Organochlorines as Precursors of 3Monochloropropandiol Esters in Vegetable Oils Soon Huat Tiong, Norliza Saparin, Huey Fang Teh, Theresa Lee Mei Ng, Mohd Zairey bin Md. Zain, Bee Keat Neoh, Ahmadilfitri Md Noor, Chin Ping Tan, Oi-Ming Lai, and David R. Appleton J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04995 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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Journal of Agricultural and Food Chemistry

Natural Organochlorines as Precursors of 3-Monochloropropandiol Esters in Vegetable Oils Soon Huat Tiong,*†‡ Norliza Saparin,§ Huey Fang Teh,† Theresa Lee Mei Ng,† Mohd Zairey bin Md. Zain,† Bee Keat Neoh,† Ahmadilfitri Md Noor,§ Chin Ping Tan,‡ Oi Ming Lai,∥ David Ross Appleton†

CORRESPONDING AUTHOR FOOT NOTE * To whom correspondence should be addressed Tel.: +(603) 8942 2641 E-mail: [email protected] Current address: †

Sime Darby Technology Centre Sdn Bhd, 1st Floor, Block B, UPM-MTDC Technology Centre III, Lebuh Silikon, 43400 Serdang, Selangor, Malaysia

‡

Faculty of Food Science and Technology, Universiti Putra Malaysia, Jalan UPM, 43400 Serdang, Selangor, Malaysia

§

Sime Darby Research Sdn Bhd, Lot 2664, Jalan Pulau Carey, 42960 Carey Island, Selangor, Malaysia

∥

Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Jalan UPM, 43400 Serdang, Selangor, Malaysia

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ABSTRACT

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During high-temperature refining of vegetable oils, 3-monochloropropandiol (3-MCPD)

3

esters, possible carcinogens, are formed from acylglycerol in the presence of a chloride

4

source. To investigate organochlorine compounds in vegetable oils as possible

5

precursors for 3-MCPD esters, we tested crude palm, soybean, rapeseed, sunflower,

6

corn, coconut and olive oils for the presence of organochlorine compounds. Having

7

found them in all vegetable oils tested, we focused subsequent study on oil palm

8

products. Analysis of chlorine isotope mass pattern exhibited in high-resolution mass

9

spectrometry enabled organochlorine compounds identification in crude palm oils as

10

constituents of wax esters, fatty acid, diacylglycerols, and sphingolipids, which are

11

produced endogenously in oil palm mesocarp throughout ripening. Analysis of thermal

12

decomposition and changes during refining suggested that these naturally present

13

organochlorine compounds in palm oils and perhaps in other vegetable oils are

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precursors of 3-MCPD esters. Enrichment and dose-response showed a linear

15

relationship to 3-MCPD ester formation and indicated that the sphingolipid-based

16

organochlorine compounds are the most active precursors of 3-MCPD esters.

17

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KEYWORDS

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3-MCPD esters, wax ester, fatty acid, diacylglycerol, sphingolipid, palm oil, chlorine

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isotopic mass patterns, high-resolution mass spectrometry

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INTRODUCTION

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3-monochloropropandiol (3-MCPD) esters are processing contaminants generated

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during the deodorization of vegetable oil in the refinery.1,

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mg/kg have been reported from coconut, corn and palm oils3 that were mostly used to

27

produce food products. 3-MCPD esters in foods consumed undergo almost full

28

hydrolysis in the human digestive system, yielding free 3-MCPD,4

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categorized by the European Scientific Committee in 2001 as a non-genotoxic threshold

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carcinogen and by the International Agency for Research on Cancer as compounds

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possibly carcinogenic to humans.5, 6 As has been reported from an in vivo rat model

32

study, the introduction of free 3-MCPD led to progressive nephrotoxicity in both male

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and female rats as well as testicular toxicity and mammary glandular hyperplasia only in

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male rats.7

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Most vegetable oils except extra virgin or virgin vegetable oils would be subjected to

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refining, commonly by physical approach that include degumming, bleaching and lastly

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almost exclusive formation of 3-MCPD esters during deodorization.8 Therefore, pre-

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deodorization treatment of vegetable oils would affect the formation of 3-MCPD esters

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such as water degumming and bleaching had reduced the formation of 3-MCPD esters

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as compared with crude vegetable oils without treatment after deodorization.9-11 The

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formation of 3-MCPD esters in vegetable oils during deodorization were believed to

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undergone via two established mechanisms: free radical addition and nucleophilic SN2

43

substitution, involving either chlorine radicals or ions generated from a source with

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acylglycerol.12, 13

2

Concentrations of > 5.0

which was



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Addition of a range of chlorinated compounds, both inorganic and organic, into

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vegetable oil samples have been reported to result in higher 3-MCPD ester formation

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upon deodorization.14-16

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inorganic chloride salts, such as sodium chloride was only 32 % and decreased to only

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6% with lower dosages.16 Free inorganic chlorine has also been reported to represent

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less than 1 mg/kg out of 2-3 mg/kg of total chlorine content in crude or refined palm oil,2

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while their amount in bleached palm oils was not well correlated with levels of 3-MCPD

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esters formed during refining.17 Water washing of crude palm oils to eliminate water

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soluble chloride sources before refining reduces, but not eliminates 3-MCPD ester

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formation.1,21 Therefore, it could be inferred that the most persistent precursors of 3-

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MCPD esters in edible oils are predominantly oil soluble, or at most partially soluble

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water organochlorine compounds.

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Organochlorine compounds are more lipophilic and difficult to remove or eliminate than

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inorganic chlorinated compounds, and are therefore the greatest challenge to mitigate

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the formation of 3-MCPD esters in vegetable oils. Crude palm oil (CPO) analysis using

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high-resolution mass spectrometry (HRMS) that effectively resolves the characteristic

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chlorine isotopic mass pattern was reported by Nagy et al. in 2011. The structural

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similarity of organochlorine compounds to lipids and their presence in oil palm

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mesocarp supported the assumption that they were produced endogenously in oil palm

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fruits18 and furthermore implied their existence in other edible oils.22 Therefore, this

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study was designed to confirm the possibility that natural organochlorine compounds in

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palm oil are precursors of 3-MCPD esters, and determine if these are ubiquitous in all

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vegetable oils.

The 3-MCPD ester conversion rate from the introduced


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MATERIALS AND METHODS Reagents and chemicals

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3-chloropropane-1,2-dipalmitoyl

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dipalmitoyl ester (Purity ≥98%) were purchased from Toronto Research Chemicals Inc.

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(North York, Canada). Silica gel (60 mesh) was purchased from Merck (Darmstadt,

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Germany). All reagents and solvents used for this study were analytical reagent grade

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except in the HRMS analysis, which used only mass spectrometry grade solvent.

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ester

and

pentadeuterated

3-chloropropane-1,2-

Samples

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Samples of crude palm oils were obtained from a palm oil mill located in central

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peninsular Malaysia (Sime Darby Plantation, Malaysia) was used for total chlorine

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content correlation with 3-MCPD esters upon refining (n=110) and subsequent

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experiment include organochlorine analysis as crude or during refining and silica

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column chromatography (n=2). Samples of crude soybean, rapeseed, and coconut oils

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(n=2 for each type of oil) were obtained from a European supplier (Sime Darby Unimills,

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Netherlands). A sample of crude sunflower oil (n=1) was obtained from a South African

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supplier (Sime Darby Hudson & Knight, South Africa). Crude corn oil samples (n=2)

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were obtained from a supplier in China. Extra virgin olive oil (n=1) produced in Spain

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was purchased from a local retailer. Oil palm fruits from six trees were collected from a

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Malaysian estate (Sime Darby Plantation, Malaysia).

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Total chlorine content analysis



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Oil sample was transferred (50 ± 5 mg) into the sample holder for total chlorine analyzer

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(Mitsubishi Chemicals NSX-2100 series) and weighed. The samples were first

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combusted under argon gas flow and later under oxygen gas flow to completion. HCl

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gas produced from the combustion was dehydrated with argon and oxygen gas before

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being directed into a titration cell with acetic acid (85%, w/w) as the electrolyte. HCl

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was automatically titrated by silver ions generated colourmetrically. The amount of

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chlorine was calculated from the quantity of electricity required for the generation of the

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silver ions used in titration. The LOQ and LOD of total chlorine analysis were 0.01 and

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0.003 µg of absolute chlorine respectively.

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Vegetable oil refining

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Crude vegetable oil was weighed (200-300 g) and poured into a round bottom flask for

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the physical refining process that includes degumming, bleaching, and deodorization.

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The crude oil was degummed with addition of 1% (w/w) phosphoric acid aqueous

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solution (85%, w/v; Nylex Bhd., Shah Alam, Malaysia) or 0.1% (v/w) citric acid aqueous

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solution (50%, w/v) with stirring for 15 min at 90°C at atmospheric pressure.

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Subsequently, the oil was bleached with the addition of 0.7% w/w of acid-activated

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bleaching clay (Taiko Bleaching Earth Sdn Bhd, Parit Buntar, Malaysia) with stirring for

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30 minutes as previously. Finally, the deodorization was performed at 260°C under

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vacuum (< 5 mbar) with steam sparging for 90 min. All refined vegetable oils were then

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subjected to 3-MCPD analysis.

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3-MCPD esters analysis


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The 3-MCPD esters and related compounds in the refined vegetable oil were analyzed

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as in Ermacora & Hrncirik.23 About 100 (± 5) mg of oil was spiked with an internal

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standard (PP-3-MCPD-d5) in 1 mL of tetrahydrofuran. It was further subjected to

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hydrolysis with 1.8 mL of sulphuric acid in methanol (1.8%, v/v) for 16 h at 40°C. The

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hydrolysis reaction was stopped by addition of saturated sodium bicarbonate followed

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by evaporation of the organic solvent under a nitrogen stream. The fatty acid methyl

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ester was removed by n-heptane in a solvent partition consisting of 2 mL sodium

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sulphate solution (20% w/v). The free 3-MCPD in aqueous was further derivatized by

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addition of 250 µL of phenyl boronic acid (PBA)- saturated solution (acetone/water, 19:1,

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v/v) and incubated for 5 min under ultrasonication at room temperature. The PBA

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derivative was extracted with n-heptane followed by drying under evaporation before

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reconstituting in 400 µL of n-heptane for gas chromatography-mass spectrometry

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analysis. The repeatability and reproducibility of the 3-MCPD esters analysis used

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were >90% with recovery of >94% and 0.02 mg/kg as the LOD.

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Organochlorine analysis by LC-HRMS

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The organochlorine substances in the oil samples were analyzed by a liquid

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chromatography high-resolution mass spectrometry (LC-HRMS) system comprising

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Dionex UltiMate 3000 UHPLC systems coupled with Q-Exactive™ Plus Orbitrap™ Mass

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Spectrometer (Thermo Fisher Scientific Inc, Massachusetts, United States) with the

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mass resolution set at 70,000.

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Oil from each sample was weighed (50.0 ± 1.0 mg) to prepare for LC-HRMS analysis.

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Each sample was dissolved in 500 µL of 60:40 (v/v) acetonitrile and isopropanol solvent

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mixture. These samples were then homogenized by vortex for 30 seconds and mixed by 7 ACS Paragon Plus Environment


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thermomixer at 40°C and 1400 rpm for 30 min. Next, these samples were centrifuged at

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15°C and 8000 rpm for 10 min to obtain a clear aliquot with undissolved substance

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settled down at bottom of the tube. The clear aliquot was transferred to sample vials for

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LC-HRMS analysis.

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The LC system was equipped with ACQUITY UPLC BEH C8 Column, 2.1 mm X 100

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mm with VanGuard Pre-column guard, 2.1 X 5 mm (Waters, 130Å, 1.7 µm).

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Temperatures of 55°C and 15°C were set for the column oven and the sample tray,

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respectively. The mobile phase used was 40:60 (v/v) acetonitrile:water for A and 90:10

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(v/v) isopropanol:acetonitrile for B, both consisting of 10 mM ammonium acetate as a

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buffer in mobile phase A and B flowing at the rate of 260 µL/min. The LC mobile phase

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profile for analysis at 0-1.5 min was 32% B followed by increasing the gradient of B to

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45% at 4.0 min, to 52% at 5.0 mins, to 58% at 8.0 min, to 66% at 11.0 min, to 70% at

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14.0 min, to 75% at 18.0 min and to 97% at 21.0 to 25.0 min. Lastly, the LC was re-

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equilibrated to 32% of B at 25.1 to 30.0 min before initiating another analysis.

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Identification and quantification experiments were conducted under a 60°C heated

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negative ESI mode with a spray and capillary voltage of 4.5 kV and -34 V, respectively,

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with capillary temperature of 275°C. Nitrogen was used as the sheath and auxiliary

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gases with flow rate at 38 and 12 arbitrary units, respectively. The tube lens was

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adjusted to -133 V. Other parameters were the typical values optimized during

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calibration. A mass fragmentation experiment was conducted with CID set at 35 kV.

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LC-HRMS data were examined for the presence of chlorinated compounds by chlorine

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pattern trace and scoring using Compound Discoverer 2.0 software (Thermo Fisher

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Scientific Inc, Massachusetts, United States). A mass tolerance of 0.002 dalton was 8 ACS Paragon Plus Environment

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specified; 10% intensity threshold was set for both chlorine pattern trace and scoring.

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Chromatographic peak areas of the identified candidate were extracted in ±0.001 m/z

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windows by Xcalibur 4.0 Quanbrowser software (Thermo Fisher Scientific Inc,

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Massachusetts, United States).

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Extraction of oil palm mesocarp

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Three oil palm fruits from a single bunch at 14 to 22 weeks after pollination (WAP) from

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six trees were sampled. The palm fruits sampled were immediately frozen in liquid

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nitrogen or in a -80°C freezer until further preparation. The oil palm mesocarp was

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separated from the fruits and further pulverized to the fine powder under liquid nitrogen.

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Fine mesocarp powder samples (100 ± 5 mg) were used for extraction according to

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modify method published.24 Briefly, the fine mesocarp powder sample was extracted

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with 3 mL of isopropanol containing 0.05% (w/v) butylated hydroxytoluene and

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homogenized

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chloroform:water:methanol (1:1:1, v/v/v) after cooling to room temperature. The mixture

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of the solvent with mesocarp fine powder was homogenized by vortex before

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subsequent mixing at 250 rpm and incubation for 1 h at 60°C. Then, the mixture was

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centrifuged and the supernatant collected while residue was subjected to re-extraction

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with 6 mL of chloroform: water (1:1, v/v). The second supernatant obtained was

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combined with the first to be further centrifuged, resulting in an aqueous layer on the top.

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The aqueous layer was discarded, and the bottom layer of non-polar constituents was

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then cleaned with 5 mL of potassium chloride (1M). The clean non-polar fraction was

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dried under vacuum and reconstituted with 500 µL of 60:40 (v/v) acetonitrile:

at

75°C for 15 minutes, followed

by addition

of

6 mL of



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isopropanol solvent mixture following the sample preparation for organochlorine

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analysis by LC-HRMS described above.

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Silica column chromatography of crude palm oil and thermal treatment

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Fractionation of organochlorine compounds through silica gel column was achieved with

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a slight modification of the oil purification method published.25 About 250 (± 5) g of

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crude palm oil (CPO-Feed) was dissolved in 200 mL petroleum ether for separation on

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300 g silica gel column. The silica gel column was eluted with solvents as a mobile

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phase as described in Table 1. The CPO-Feed, fractions (F1-5) obtained and F1 model

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oil with different F5 dosage (10, 30, 50, 70, 90%) were subjected to thermal treatment

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according to a modified version of the published method.25 Briefly, 2000 ± 100 mg oil

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was measured into a 50 mL conical flask and placed in a temperature-controlled oven

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under vacuum for thermal treatment. The sample in the oven was heated at 200°C with

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a vacuum of 200°C) and effective pH state of the oil.

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Effects of total chlorine content in CPO on 3-MCPD esters in Refined

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Bleached Deodorized Palm Oil (RBDPO) 11 ACS Paragon Plus Environment


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Total chlorine was measured in CPO samples using a total chlorine analyzer based on

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the oxidative combustion and microcoulometry principle and was compared with 3-

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MCPD ester concentrations formed during refining. 3-MCPD esters in the RBDPO

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exhibited a statistically significant positive correlation (p < 0.001, r > 0.85) with total

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chlorine content in CPO (n=110) in a regression model evaluation (Figure 1), suggesting

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chlorinated compounds as the precursors for 3-MCPD formation. Our results supported

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previous findings where addition of chlorinated compounds to crude vegetable oils

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resulted in higher 3-MCPD formation.14-16

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The analysis of total chlorine content is a measurement of the products from chlorine

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combustion, inclusive of free inorganic chloride and bound organochlorine compounds,

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of which both could be the chloride source for 3-MCPD ester formation. Chlorinated

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precursors of 3-MCPD would need to be present in the oil during deodorization,21 and

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therefore must be oil soluble and not removed significantly during degumming and

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bleaching. While inorganic chlorides such as iron (II) or (III) chloride have been reported

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in CPO,18 iron content has been shown to reduce significantly during degumming and

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bleaching.10,

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concentrations of less than 0.40 ppm after degumming with phosphoric acid at 0.06 wt%

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of CPO and bleaching with 1.0% clay used.19 More than 200 organochlorine compounds

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have been discovered in CPO18 and are persistent in the oil until deodorization,

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supporting their potential role as the prominent chlorinated precursors of 3-MCPD

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esters. Therefore, our study focused on the presence of organochlorines in vegetable

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oils to evaluate their contribution to the formation of 3-MCPD esters.

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20

About 80-90% of iron content in CPO was reduced with final

Organochlorine in Crude Vegetable Oils and their Origin 12 ACS Paragon Plus Environment

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To further explore the presence and nature of organochlorine compounds in vegetable

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oils, we profiled extra virgin olive oil, crude soybean oils, crude rapeseed oils, crude

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sunflower oil, crude corn oils, crude coconut oils and crude palm oils. Organochlorine

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compounds in crude vegetable oils were evaluated through the chlorine characteristic

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isotope mass defect and ratio exhibited in HRMS analysis developed and used to report

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such compounds from CPO.18 In addition to the organochlorine compounds that have

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previously been discovered in CPO, other newly discovered organochlorine compounds

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were identified and described (Table 2). A range of 100-150 compounds was

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discovered as potential organochlorines in all the crude vegetable oils, ranging from

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seed oils (soybean, rapeseed, sunflower, corn, and coconut) to mesocarp/flesh oils (oil

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palm and olive), that were evaluated in this study.

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Several of the most abundant organochlorine compounds identified were the wax ester,

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fatty acid, diacylglycerol and sphingolipid derivatives based on chemical formulae

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generated from exact mass and mass fragmentation observed in HRMS analysis. The

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most abundant organochlorine with exact mass for [M-H]- at 391.26184 m/z (OC 391,

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Table 2) suggested C21H40O4Cl- as the most probable chemical formula. Hence, we

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identified OC 391 as dihydrochloropentyl palmitate wax ester (Figure 3) with the support

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of mass fragmentation that showed a palmitoyl fragment. Although all literature on wax

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ester mass fragmentation have been on positive ionization, it also supports the loss of

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acyl fragment from the ester observed in our negative MS fragmentation.27,

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Organochlorine with [M-H]- of 389.24622 m/z was also designated a chlorinated wax

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ester based on the mass difference of 2.01555 ± 0.00011 m/z with OC 391, which

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suggests desaturation by two hydrogens (theoretical: 2.01510 m/z). This was confirmed

28



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by mass fragmentation of OC 389 showing 253.21716 m/z. The position of the chlorine

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atom and unsaturation could not be confirmed.

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The organochlorine with [M-H]- of 347.19901 m/z (OC 347, Table 2) with the suggested

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chemical formula of C18H43O4Cl- with two degrees of unsaturation was assumed to

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undergo neutral loss of H2O and HCl molecule during mass fragmentation ([M-H-H2O-

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HCl]- 293.21194 m/z, suggesting an oleate-related fragment. Therefore, we identified

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OC 347 as dihydrochloro-oleic acid (Figure 3). The exact mass of OC 659 suggested

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the chemical formula of C37H68O7Cl- with three degrees of unsaturation. Two prominent

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mass fragments were observed, [R1COO]- (255.23253 m/z; Fragment 1) and [R2COO-

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HCl-H2O]- (293.21209 m/z; Fragment 2), suggesting palmitate- and oleate-related

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fragments. The chemical formula and mass fragmentation were evidence for proposing

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them to be chlorinated diacylglycerol. Previous mass fragmentation studies of

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acylglycerol were conducted in positive mode.29,

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glycerophosphoethanolamine, which possessed similar stearic configuration as

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diacylglycerol, showed that the preferential RCOO- fragment was from sn2 rather than

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from sn1.31 Therefore, diacylglycerol would exhibit a similar pattern of mass fragments

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and supported the identification of OC 659 Fragment 1 at sn2 of a glycerol backbone.

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As a result, we identified OC 659 as 18:1(OH & Cl)/16:0 diacylglycerol (Figure 3). OC

286

683 showed mass characteristics similar to those of OC 659, which led us to identify it

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as 18:1(OH & Cl)/18:3 diacylglycerol.

30

Mass fragmentation of diacyl

288 289

OC 718 has been previously identified as a sphingolipid based on its mass

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fragmentation pattern that suggests the similarity of its structure with that of


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phytosphingosine.18 Phytosphingosine is a member of the ceramide class of

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sphingolipids present in living organisms, including in plants such as Arabidopsis.32-34

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The biosynthesis of sphingolipids must involve acylation of amine in sphinganine by

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ceramide synthase to give rise to N-acyl bonding (Figure 2), as seen in ceramide.35

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Ceramide readily undergoes hydroxylation of acyl amide at α,36 2 and 3 positions with

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about 2-3% of 2,3-dihydroxylated fatty acid amides that have been detected in plants.37,

297

38

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dihydroceramide (d18:0-24:0, Figure 2) related compounds based on reported

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biosynthetic pathways of sphingolipid that are in accordance with the mass fragments

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reported and observed in our study.18 We further inferred that OC 600, 614, 700 and

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776 were sphingolipid based on the chemical formula established with < 3 ppm

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deviation containing a single nitrogen generated from exact mass and on the similarity

303

of its [M-HCl]- fragmentation to that seen in OC 718. The other organochlorines with

304

insufficient mass fragmentation for identification were grouped as unknown (Table 2).

Therefore, we propose a revised structure of OC 718 and identified OC 718 as

305 306

Sphingolipid organochlorines showed higher content in crude vegetable oils with > 0.5

307

mg/kg of 3-MCPD esters formed after refining (except for sunflower oil) (Table 2). The

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organochlorine compounds in crude palm oils have been previously found to be

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endogenously produced by oil palm based on their presence in oil palm fruit mesocarp

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and their lipid-like structure.18 Therefore, our initial investigation strongly suggests

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sphingolipid organochlorines to be potential chlorinated precursors of 3-MCPD esters in

312

vegetable oils since they appear to be ubiquitous in all plant oils. Hence, we evaluated

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oil palm mesocarp at different maturation and ripeness stages for the presence of



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organochlorine compounds related to with lipid biosynthesis to prove organochlorine as

315

naturally synthesized compounds in fruits.

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Organochlorine compounds were found to be present in oil palm mesocarp as early as

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14 weeks after pollination (WAP) before the initiation of lipid biosynthesis during 15-16

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WAP. Contents of wax esters and sphingolipid organochlorine compounds increased

319

with increasing oil content per dry matter of oil palm mesocarp (Figure 3).39 Therefore,

320

we have confirmed the previous finding of organochlorine compounds as lipid-related

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compounds produced endogenously by oil palm through lipid biosynthesis.18 The fatty

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acid diacylglycerol as well as unknown organochlorines showed incremental abundance

323

from 14 WAP, achieving maximum abundance between 17 and 21 WAP before

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decreasing as the palm fruit becomes ripe at 22 WAP (Figure 3). Therefore, our study

325

suggested that the formation of organochlorine compounds is coincident with lipid

326

biosynthesis.

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Organochlorine as precursors of 3-MCPD esters during deodorization

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3-MCPD esters and derivatives are formed almost exclusively during deodorization.8, 40

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Changes in organochlorine compound concentrations during deodorization and whether

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these compounds are retained in the palm fatty acid distillate (PFAD) would enable

331

identification of potential reactive organochlorine compounds that may provide chlorine

332

for 3-MCPD ester formation. Most of the identified organochlorine compounds showed

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significant (p < 0.10, Figure 4) reduction after deodorization except for OC 389 (p>0.10,

334

Figure 4). All of these organochlorines appeared to have decomposed, with the

335

exception of the fatty acid organochlorine (OC 347) that appeared at high concentration

336

in the distillate (PFAD). Therefore, our findings indicated that most organochlorine 16 ACS Paragon Plus Environment

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compounds identified except fatty acid organochlorine compound were potential

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reactive organochlorine as precursors of 3-MCPD esters which will be further evaluated

339

in CPO silica column chromatography fraction study.

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Silica column chromatography of crude palm oil (CPO-Feed), resulted in five fractions

341

(F1-5) being obtained with wax esters, diacylglycerol and sphingolipid organochlorine

342

compounds enriched or fractionated into 4th and 5th fractions (F4 & F5, Figure 5).

343

Fractionation was more focused on separating the polar organochlorine compounds

344

from acylglycerols compared to previous studies involving silica fractionation25 with an

345

additional stronger solvent fraction (F5) of 20% methanol and almost full recovery (>

346

98%) in terms of weight. Thermal treatment of F1-5 at 200 °C for 6 h resulted in the

347

formation of high amounts of 3-MCPD esters only in F5 (19.00 mg/kg), with lesser in F4

348

(2.80 mg/kg) and negligible amounts in F1-3 (0.98) to F5 dosing (Figure 6),

358

this further confirmed organochlorine compounds as 3-MCPD ester precursors. Our

359

results suggested that the possible action of organochlorine under deodorization to form



Page 18 of 30

360

3-MCPD esters and derivatives with acylglycerol arises during decomposition at high

361

temperature, which liberates HCl which then plays a role as the chloride source.14

362

Reviewing the difference in behavior and property of wax esters, diacylglycerol and

363

sphingolipid organochlorine exhibited during vegetable oil refining and their ability to

364

liberate HCl during mass spectrometry fragmentation experiments, we identified

365

differences in reactivity of organochlorine compounds. The lower reduction (65%) and

371

sphingolipid (70-100%) organochlorines along with the facile loss of HCl during MS

372

fragmentation. Therefore, the higher contents of 3-MCPD esters in F4 and F5 after

373

thermal treatment compared to that formed in F1-3 was believed to be caused by the

374

presence of diacylglycerol and sphingolipid organochlorine compounds. Significantly

375

higher 3-MCPD ester formation observed in F5 compared to F4, despite having 70%

376

less relative diacylglycerol organochlorine content. Sphingolipid organochlorine

377

compounds were the most lipophilic, reduced the most during deodorization and easily

378

liberated HCl during MS fragmentation and are therefore likely to be the most reactive

379

organochlorine compounds as precursors of 3-MCPD esters in palm oil.

380

We confirmed that organochlorine compounds present in crude oils are the main

381

contributor of 3-MCPD esters and that these compounds are present in differing

382

degrees in all vegetable oils. We then used mass fragment data to assign 18 ACS Paragon Plus Environment

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383

organochlorines into distinct classes and investigated through mass fragmentation and

384

profiling during high-temperature vacuum distillation their propensity to degrade with

385

loss of HCl. We found sphingolipid organochlorines to be the most labile chloride source

386

and under controlled dosing yielded linear formation of 3-MCPD esters. This is highly

387

suggestive of their important role in the formation of 3-MCPD esters. Future studies

388

could investigate other factors that could influence the formation of 3-MCPD esters,

389

such as partial acylgylcerol content or oil acidity. Nevertheless, our results confirmed

390

the importance of diacylglycerol and predominantly sphingolipid organochlorine

391

compounds as precursors of 3-MCPD esters while other identified organochlorines

392

described in Table 2 were less likely to act as precursors of 3-MCPD esters. Given that

393

these compounds have now been observed to form along with oil biosynthesis in fruits,

394

attention can now turn to the timing of the formation of these compounds in the palm

395

fruit intending to reduce their production earlier in fruits or removal during processing

396

and vegetable oil production.

397

ASSOCIATED CONTENT

398

Identification of lipid (wax esters, fatty acid, diacylglycerol, sphingolipid) organochlorine

399

by accurate mass and mass fragments (Table S1 & Figure S1-S4) obtained in high

400

resolution mass spectrometry.

401

ACKNOWLEDGEMENT

402

We wish to acknowledge Thermo Scientific, Malaysia for providing their Dionex UltiMate

403

3000 UHPLC systems with Q-Exactive™ Plus Orbitrap™ Mass Spectrometer and

404

Compound Discoverer 2.0 software for this study analysis. We also wish to



Page 20 of 30

405

acknowledge Mr. Wong Yick Ching and Ms. Koo Ka Loo from Sime Darby Technology

406

Centre, Malaysia, for collecting the oil palm fruit samples and for conducting the

407

extraction. The 3-MCPD analysis was conducted by Ms. Maizatul Putri Ahmad Sabri

408

from Sime Darby Research Sdn. Bhd., Malaysia. We also like to thank Alena Sanusi for

409

editorial advice.

410

NOTES

411

The authors declare no conflict of interest.

412

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FIGURE CAPTIONS Figure 1: Regression of 3-MCPD esters in RBDPO relative to total chlorine content in CPO (n = 110) Figure 2: Putative structure of OC 391, 347, 659 and 718 based on HRMS exact mass and mass fragmentation; unconfirmed substitutions are bolded. Figure 3: Oil content and organochlorine compounds in oil palm mesocarp at different maturation and ripeness from 14 to 22 WAP; data on organochlorine abundance were obtained from analysis of extract from standardized mesocarp (100 mg). Figure 4: Organochlorine compounds in bleached palm oil (bleached) that significantly (p

Natural Organochlorines as Precursors of 3 ... - ACS Publications

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