Natural Organochlorines as Precursors of 3-Monochloropropanediol

Subscriber access provided by Gothenburg University Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

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

1 ACS Paragon Plus Environment


Page 2 of 30

1

ABSTRACT

2

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

14

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

18

KEYWORDS

19

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

20

isotopic mass patterns, high-resolution mass spectrometry

21

22 2 ACS Paragon Plus Environment

Page 3 of 30


23

INTRODUCTION

24

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

25

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

26

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

29

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

30

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

31

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

33

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

34

male rats.7

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

acylglycerol.12, 13

2

Concentrations of > 5.0

which was



Page 4 of 30

45

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

46

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

47

upon deodorization.14-16

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

water organochlorine compounds.

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

vegetable oils.

The 3-MCPD ester conversion rate from the introduced


Page 5 of 30


68

69

70

MATERIALS AND METHODS Reagents and chemicals

71

3-chloropropane-1,2-dipalmitoyl

72

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

73

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

74

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

75

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

76

ester

and

pentadeuterated

3-chloropropane-1,2-

Samples

77

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

78

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

79

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

80

experiment include organochlorine analysis as crude or during refining and silica

81

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

82

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

83

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

84

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

85

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

86

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

87

Malaysian estate (Sime Darby Plantation, Malaysia).

88

Total chlorine content analysis



Page 6 of 30

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

0.003 µg of absolute chlorine respectively.

98

Vegetable oil refining

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

subjected to 3-MCPD analysis.

109

3-MCPD esters analysis


Page 7 of 30


110

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

111

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

112

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

113

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

114

hydrolysis reaction was stopped by addition of saturated sodium bicarbonate followed

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

Organochlorine analysis by LC-HRMS

125

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

126

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

127

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

128

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

129

mass resolution set at 70,000.

130

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

131

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

132

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


Page 8 of 30

133

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

134

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

135

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

136

LC-HRMS analysis.

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

Page 9 of 30


156

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

157

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

158

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

159

Massachusetts, United States).

160

Extraction of oil palm mesocarp

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

homogenized

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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



Page 10 of 30

178

isopropanol solvent mixture following the sample preparation for organochlorine

179

analysis by LC-HRMS described above.

180

Silica column chromatography of crude palm oil and thermal treatment

181

Fractionation of organochlorine compounds through silica gel column was achieved with

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

220

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

221

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


Page 12 of 30

222

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

223

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

224

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

225

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

226

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

227

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

228

previous findings where addition of chlorinated compounds to crude vegetable oils

229

resulted in higher 3-MCPD formation.14-16

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

bleaching.10,

238

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

239

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

240

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

241

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

242

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

243

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

244

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

Page 13 of 30


245

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

246

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

247

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

248

compounds in crude vegetable oils were evaluated through the chlorine characteristic

249

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

250

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

251

previously been discovered in CPO, other newly discovered organochlorine compounds

252

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

253

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

254

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

255

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

256

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

257

fatty acid, diacylglycerol and sphingolipid derivatives based on chemical formulae

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

28



Page 14 of 30

268

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

269

atom and unsaturation could not be confirmed.

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

them to be chlorinated diacylglycerol. Previous mass fragmentation studies of

280

acylglycerol were conducted in positive mode.29,

281

glycerophosphoethanolamine, which possessed similar stearic configuration as

282

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

283

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

284

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

285

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

287

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

290

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


Page 15 of 30


291

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

292

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

293

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

294

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

295

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

296

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

297

38

298

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

299

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

300

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

301

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

302

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

308

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

309

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

310

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

311

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

313

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



Page 16 of 30

314

organochlorine compounds related to with lipid biosynthesis to prove organochlorine as

315

naturally synthesized compounds in fruits.

316

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

317

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

318

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

321

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

322

acid diacylglycerol as well as unknown organochlorines showed incremental abundance

323

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

324

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.

327

Organochlorine as precursors of 3-MCPD esters during deodorization

328

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

329

Changes in organochlorine compound concentrations during deodorization and whether

330

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

333

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

Page 17 of 30


337

compounds identified except fatty acid organochlorine compound were potential

338

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

339

in CPO silica column chromatography fraction study.

340

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

Page 19 of 30


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

REFERENCES

413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441

1.

2.

3.

4.

5. 6.

7.

8. 9.

10.

Matthäus, B.; Pudel, F.; Fehling, P.; Vosmann, K.; Freudenstein, A., Strategies for the reduction of 3‐MCPD esters and related compounds in vegetable oils. Eur. J. Lipid Sci. Technol. 2011, 113, 380-386. Franke, K.; Strijowski, U.; Fleck, G.; Pudel, F., Influence of chemical refining process and oil type on bound 3-chloro-1, 2-propanediol contents in palm oil and rapeseed oil. LWT-Food Sci. Technol. 2009, 42, 1751-1754. Matthäus, B.; Pudel, F., Mitigation of 3‐MCPD and glycidyl esters within the production chain of vegetable oils especially palm oil. Lipid Technol. 2013, 25, 151-155. Abraham, K.; Appel, K. E.; Berger-Preiss, E.; Apel, E.; Gerling, S.; Mielke, H.; Creutzenberg, O.; Lampen, A., Relative oral bioavailability of 3-MCPD from 3MCPD fatty acid esters in rats. Arch. Toxicol. 2013, 87, 649-659. European Commission, Opinion of the Scientific Committee on Food on 3Monochloro-propane-1,2-diol (3-MCPD) Updating the SCF Opinion of 1994. 2001. International Agency for Reasearch on Cancer, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: List of Classifications. http://monographs.iarc.fr/ENG/Classification/latest_classif.php (accessed Nov 27, 2017). Cho, W.-S.; Han, B. S.; Nam, K. T.; Park, K.; Choi, M.; Kim, S. H.; Jeong, J.; Jang, D. D., Carcinogenicity study of 3-monochloropropane-1, 2-diol in Sprague– Dawley rats. Food Chem. Toxicol 2008, 46, 3172-3177. Weißhaar, R., 3‐MCPD‐esters in edible fats and oils–a new and worldwide problem. Eur. J. Lipid Sci. Technol. 2008, 110, 671-672. Pudel, F.; Benecke, P.; Fehling, P.; Freudenstein, A.; Matthäus, B.; Schwaf, A., On the necessity of edible oil refining and possible sources of 3‐MCPD and glycidyl esters. Eur. J. Lipid Sci. Technol. 2011, 113, 368-373. Ramli, M. R.; Siew, W. L.; Ibrahim, N. A.; Hussein, R.; Kuntom, A.; Razak, R. A. A.; Nesaretnam, K., Effects of degumming and bleaching on 3-MCPD esters formation during physical refining. J. Am. Oil Chem. Soc. 2011, 88, 1839-1844. 20 ACS Paragon Plus Environment

Page 21 of 30

442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487


11.

12.

13.

14.

15.

16.

17. 18.

19. 20. 21.

22.

23.

24.

25.

26. 27.

Zulkurnain, M.; Lai, O. M.; Latip, R. A.; Nehdi, I. A.; Ling, T. C.; Tan, C. P., The effects of physical refining on the formation of 3-monochloropropane-1, 2-diol esters in relation to palm oil minor components. Food Chem. 2012, 135, 799-805. Zhang, X.; Gao, B.; Qin, F.; Shi, H.; Jiang, Y.; Xu, X.; Yu, L., Free radical mediated formation of 3-monochloropropanediol (3-MCPD) fatty acid diesters. J. Agric. Food Chem. 2013, 61, 2548-2555. Rahn, A. K. K.; Yaylayan, V. A., What do we know about the molecular mechanism of 3‐MCPD ester formation? Eur. J. Lipid Sci. Technol. 2011, 113, 323-329. Destaillats, F.; Craft, B. D.; Sandoz, L.; Nagy, K., Formation mechanisms of monochloropropanediol (MCPD) fatty acid diesters in refined palm (Elaeis guineensis) oil and related fractions. Food Addit. Contam. 2012, 29, 29-37. Shimizu, M.; Weitkamp, P.; Vosmann, K.; Matthäus, B., Influence of chloride and glycidyl‐ester on the generation of 3‐MCPD‐and glycidyl‐esters. Eur. J. Lipid Sci. Technol. 2013, 115, 735-739. Freudenstein, A.; Weking, J.; Matthäus, B., Influence of precursors on the formation of 3‐MCPD and glycidyl esters in a model oil under simulated deodorization conditions. Eur. J. Lipid Sci. Technol. 2013, 115, 286-294. Hrncirik, K.; van Duijn, G., An initial study on the formation of 3‐MCPD esters during oil refining. Eur. J. Lipid Sci. Technol. 2011, 113, 374-379. Nagy, K.; Sandoz, L.; Craft, B.; Destaillats, F., Mass-defect filtering of isotope signatures to reveal the source of chlorinated palm oil contaminants. Food Addit. Contam. 2011, 28, 1492-1500. Puah, C. W.; Choo, Y. M.; Ma, A. N.; Chuah, C. H., Degumming and bleaching: effect on selected constituents of palm oil. J. Oil Palm Res. 2004, 16, 57-63. Morad, N. A.; Aziz, M. A.; Zin, R. M. Process design in degumming and bleaching of palm oil. Universiti Teknologi Malaysia, 2006. Matthäus, B., Organic or not organic–that is the question: How the knowledge about the origin of chlorinated compounds can help to reduce formation of 3‐ MCPD esters. Eur. J. Lipid Sci. Technol. 2012, 114, 1333-1334. Brian D. Craft; Destaillats, F., Formation Mechanisms. In Processing contaminants in edible oils: MCPD and glycidyl esters, MacMahon, S., Ed. Elsevier: 2015. Ermacora, A.; Hrncirik, K., A novel method for simultaneous monitoring of 2MCPD, 3-MCPD and glycidyl esters in oils and fats. J. Am. Oil Chem. Soc. 2013, 90, 1-8. Bartz, R.; Li, W.-H.; Venables, B.; Zehmer, J. K.; Roth, M. R.; Welti, R.; Anderson, R. G.; Liu, P.; Chapman, K. D., Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic. J. Lipid Res. 2007, 48, 837-847. Ermacora, A.; Hrncirik, K., Influence of oil composition on the formation of fatty acid esters of 2-chloropropane-1, 3-diol (2-MCPD) and 3-chloropropane-1, 2-diol (3-MCPD) under conditions simulating oil refining. Food Chem. 2014, 161, 383389. Christie, W. W., Gas chromatography and lipids. Oily Press Ayr, Scotland, 1989. Camera, E.; Ludovici, M.; Galante, M.; Sinagra, J.-L.; Picardo, M., Comprehensive analysis of the major lipid classes in sebum by rapid resolution 21 ACS Paragon Plus Environment


488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524

28.

29.

30. 31.

32. 33.

34. 35. 36.

37. 38. s 39.

40.

Page 22 of 30

high-performance liquid chromatography and electrospray mass spectrometry. J. Lipid Res. 2010, 51, 3377-3388. Butovich, I. A.; Uchiyama, E.; McCulley, J. P., Lipids of human meibum: massspectrometric analysis and structural elucidation. J. Lipid Res. 2007, 48, 22202235. Mu, H.; Sillen, H.; Hiy, C.-E., Identification of diacylglycerols and triacylglycerols in a structured lipid sample by atmospheric pressure chemical ionization liquid chromatography/mass spectrometry. J. Am. Oil Chem. Soc. 2000, 77, 1049-1060. Byrdwell, W. C., Atmospheric pressure chemical ionization mass spectrometry for analysis of lipids. Lipids 2001, 36, 327-346. Hsu, F.-F.; Turk, J., Charge-remote and charge-driven fragmentation processes in diacyl glycerophosphoethanolamine upon low-energy collisional activation: A mechanistic proposal. J. Am. Soc. Mass Spectrom. 2000, 11, 892-899. Pata, M. O.; Hannun, Y. A.; Ng, C. K. Y., Plant sphingolipids: decoding the enigma of the Sphinx. New Phytol. 2010, 185, 611-630. Markham, J. E.; Jaworski, J. G., Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed‐phase high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 1304-1314. Spiegel, S.; Milstien, S., Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat. Rev. Mol. Cell Biol. 2003, 4, 397-407. Lynch, D. V., [15] Enzymes of sphingolipid metabolism in plants. Methods Enzymol. 2000, 311, 130-149. Kaya, K.; Ramesha, C.; Thompson, G., On the formation of alpha-hydroxy fatty acids. Evidence for a direct hydroxylation of nonhydroxy fatty acid-containing sphingolipids. J. Biol. Chem. 1984, 259, 3548-3553. Ito, S.; Ohnishi, M.; Fujino, Y., Investigation of sphingolipids in pea seeds. Agric. Biol. Chem.1985, 49, 539-540. Ohnishi, M.; Ito, S.; Fujino, Y., Structural characterization of sphingolipids in leafy tems of rice. Agric. Biol. Chem. 1985, 49, 3327-3329. Teh, H. F.; Neoh, B. K.; Hong, M. P. L.; Low, J. Y. S.; Ng, T. L. M.; Ithnin, N.; Thang, Y. M.; Mohamed, M.; Chew, F. T.; Yusof, H. M.; Kulaveerasingam, H.; Appleton, D. R., Differential Metabolite Profiles during Fruit Development in HighYielding Oil Palm Mesocarp. PloS one 2013, 8, e61344. Craft, B. D.; Nagy, K.; Sandoz, L.; Destaillats, F., Factors impacting the formation of monochloropropanediol (MCPD) fatty acid diesters during palm (Elaeis guineensis) oil production. Food Addit. Contam. 2012, 29, 354-361.

525


Page 23 of 30


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-Monochloropropanediol

Recommend Documents