Determination of 3-Monochloropropane-1, 2-diol and 2

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Determination of MCPD Esters and Glycidyl Esters by Microwave Extraction in different Foodstuffs Corinne MARC, Valerie DROUARD-PASCAREL, Cécile Retho, Patrice Janvion, and Frédéric Saltron J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00770 • Publication Date (Web): 02 May 2016 Downloaded from http://pubs.acs.org on May 12, 2016

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Journal of Agricultural and Food Chemistry

TITLE AND AUTHORSHIP

Determination of MCPD Esters and Glycidyl Esters by Microwave Extraction in different Foodstuffs

Corinne Marc*, Valérie Drouard-Pascarel, Cécile Rétho, Patrice Janvion, Frédéric Saltron

Service Commun des Laboratoires, 25 Avenue de la République ,91744 Massy, France

* [email protected]; +33169538751

ACS Paragon Plus Environment


1

ABSTRACT

2 3

This paper describes a method for the determination of MCPD (3-monochloropropane-

4

1,2-diol and 2-monochloropropane-1,3-diol) esters and glycidyl esters in various

5

foodstuffs which are isolated using a microwave extraction. The next step is based on

6

alkaline-catalyzed esters cleavage. The released glycidol is transformed into

7

monobromopranediol (MBPD). All compounds are derivatized in free diols (MCPD and

8

MBPD) with phenylboronic acid and analysed by gas chromatography – mass

9

spectrometry (GC-MS). The method was validated for oils with limit of quantitation (LOQ)

10

of 0.1 mg/kg, for chips and crisps with LOQ of 0.02 mg/kg and infant formula with LOQ

11

of 0.0025 mg/L. Recoveries of each sample were controlled by standard addition on

12

extracts before derivatization. Quantitation was performed by the addition of isotopically

13

labeled glycidyl esters and 3-MCPD esters.

14 15

KEYWORDS: 3-MCPD esters, 2-MCPD esters, glycidyl esters, microwave

16

extraction, foodstuffs, GC-MS

17 18


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19


INTRODUCTION

20 21

3-monochloropropane-1,2-diol (3-MCPD) is a compound that can be generated during

22

food preparation processes, and is available in various foods, including hydrolyzed

23

vegetable proteins, soy sauces, baked products, etc.

24

proteins and soy sauces, a maximum level of 20 µg/kg was set by the EU Regulation

25

836/20114 provides methods of sampling and analysing for the official control of levels of

26

3-MCPD in foods.

27

The first papers concerning the presence of 3-MCPD esters, were published in the

28

1980s.

29

vegetable oils and derivatives has attracted the attention of risk managers for a several

30

years. High concentrations (approximately 4 mg/kg) have been found in hydrogenated

31

fat, palm oil and solid fats used for frying.7 Thereafter, 2-monochloropropane-1,3-diol

32

esters (2-MCPD esters) and glycidyl esters have been found in oils and fats.8, 9

33

The potential hazard has recently become more clear when it was discovered that 3-

34

MCPD esters convert into 3-MCPD in the human intestine.10-14 The toxicity of 3-MCPD

35

and glycidol has been confirmed by the International Agency on Research of

36

Cancer.15,16

37

In recent years, various methods have been developed for the determination of these

38

esters in fats and oils. Initially, great difficulties have emerged due to the mutual

39

conversion of the three esters during the extraction. Several quantitation methods of

40

esters in fats and oils have already been standardized by international organizations.17-20

41

However, no methods for foodstuffs have been agreed upon to date.21 Indeed, it is

42

difficult to implement the extraction of the fat without mutual conversion of these

5, 6

1, 2

For hydrolyzed vegetable

However, the discovery of their presence in high quantities in refined



43

esters.22-24 The presence of inorganic chloride, for instance, overestimates the amount

44

of MCPD after alkaline treatment. The indirect method used was based on the fact that

45

an acidic pre-treatment eliminates glycidol and glycidyl derivatives, while the amount of

46

MCDP does not change. Glycidyl derivates were eliminated with the use of acidic

47

catalysed transesterification. Further, MCPD and glycidol could be released if no acidic

48

pre-treatment was applied due to alkaline catalysed transesterification. Methods are

49

being validated for the determination of esters in oil-based emulsions such as

50

margarines and spreads.25 Limits values are required by the recommendation

51

2014/661/EU.26

52 53

This paper presents a new method for the extraction of esters using a microwave. The

54

extraction in the microwave is most commonly used for extraction of contaminants in

55

non-food matrices (ie. sediment, water, etc).27 However, the use of this promising

56

technique is becoming common in more and more laboratories and has also recently

57

been used in the determination of PAH (Polycyclic aromatic hydrocarbon) in foodstuffs.

58 59

28

The aim of the microwave was to get a fast, efficient and economic extraction of fat in

foodstuffs including the matrices monitored by the European community.29

60 61

This paper aims to present a new method of extraction for the analysis of the MCPD

62

esters and glycidyl esters in foodstuffs. For the quantitation of the esters, the method

63

used is adopted from the work of Jan Kuhlmann and the official methods of the

64

American Oil Chemists' Society (AOCS) which have been described in detail to facilitate

65

its use in other laboratories.9.

19, 20

This method employs an indirect approach using


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66

validation data to indicate good linearity, recovery, accuracy, and low limits of detection

67

for

each

ester.

68 69

MATERIALS AND METHODS

70 71

Chemicals. 1-2-hexanediol (≥ 98%), phenylboronic acid (≥ 97%), sodium bromide and

72

ethyl acetate (free of higher boiling impurities) were bought from Sigma-Aldrich, Saint

73

Quentin Fallavier, France. Methanol, iso-octane, diethyl ether, n-heptane, anhydrous

74

sodium sulfate and orthophosphoric acid (85%) were purchased from VWR, Fontenay-

75

sous-Bois, France. Glycidyl palmitate (glycidyl ester, ≥ 98%), glycidyl palmitate d5

76

(glycidyl d5 ester, ≥ 95%, 98.8% atom) 1,3 dipalmitoyl-2-chloropropanediol (3-MCPD

77

ester, ≥ 98%), rac 1,2-bis-palmitoyl-3-chloropropanediol (2-MCPD ester, ≥ 95%) and (±)-

78

1,2-bis-hexadecanoyl-3-chloropropane-d5-diol (3-MCPD d5 ester, ≥ 98%, 98% atom)

79

were obtained from Cluzeau, Courbevoie, France (TRC Product). All solvents were

80

stored over (small amounts) of anhydrous sodium sulfate.

81 82

Reagents. A methanolic sodium hydroxide solution was prepared by dissolving 125 mg

83

of sodium hydroxide in 50 mL of methanol.

84

A sodium bromide acid solution was prepared by dissolving 30 g of NaBr in 50 mL of

85

deionized water and then acidified with the addition of 170 µL of orthophosphoric acid.

86

600 µL of this solution was used to neutralize 350 µL of the methanolic sodium

87

hydroxide solution and if necessary, the pH-value was adjusted to the acidic range.

88

The derivatization reagent (phenylboronic acid: PBA) was prepared by dissolving 200

89

mg of PBA in 10 mL of diethyl ether.



90 91

Standard Solutions. Solutions of 1,2-hexanediol were prepared in diethyl ether/ethyl

92

acetate (60/40). A solution at 0.5 µg/mL was prepared and will be used thereafter to

93

determine the recovery of the extraction of esters.

94

Standard solutions of 3-MCPD ester, 2-MCPD ester, glycidyl ester and 3-MCPD d5 ester

95

at 1 mg/mL were prepared with toluene as solvents, while a standard solution of glycidyl

96

d5 ester at 50 µg/mL was prepared with toluene as a solvent.

97

Spiking solutions were prepared by diluting stock solutions of 3-MCPD esters (55 µg/mL

98

and 5.5 µg/mL), 2-MCPD esters (55 µg/mL and 5.5 µg/mL), glycidyl ester (100 µg/mL

99

and 10 µg/mL) and 3-MCPD d5 ester (40 µg/mL), respectively, to the desired

100

concentration using toluene as a solvent.

101

All of these solutions were stored in a freezer at -18°C for six months.

102 103

Calibration Solutions. For quantitation purposes by external calibration on GC-MS

104

instrument, a calibration curve was generated by diluting in diethyl ether/ethyl acetate

105

(60/40) the unlabeled MCPD esters at concentration of 0.15, 0.5, 2, 4, 5 µg/mL and the

106

unlabeled glycidyl ester at concentration of 0.25, 1, 3, 7, 9 µg/mL. Each contained the

107

isotopically labeled 3-MCPD esters at a constant concentration of 2 µg/mL and the

108

glycidyl esters at a constant concentration of 2.5 µg/L. The entire method was then

109

applied to the calibration solutions, namely the procedure for esters cleavage and matrix

110

clean-up, and the derivatization procedure.

111 112

Sample Preparation. All extractions were performed in a Monowave 450 microwave

113

reactor (Anton Paar) with 30 mL glass vials, utilizing the integrated 24-position


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autosampler. The septa used were 22 mm EPA Septa Silicone/PTFE, 3.2mm thick. The

115

septa were changed for each extraction.

116

For oils. The sample was homogenized by stirring for several minutes, and a 100 mg

117

sample was weighted and spiked with 50 µL of the stock solution of 3-MCPD d5 ester

118

(40 µg/mL) and 50 µL of the standard solution of glycidyl d5 ester (50 µg/mL).

119

For chips and crisps. The sample was milled prior to extraction using a blixer, and the

120

appropriate weight of sample was

121

where x was the amount of sample to weigh in mg and y was the fat percentage (%) of

122

the product. The fat percentage was determinated using Röse Gottlieb method. The

123

weighted sample was added to a microwave tube and spiked with 50 µL of the stock

124

solution of 3-MCPD d5 ester (40 µg/mL) and 50 µL of the standard solution of glycidyl d5

125

ester (50 µg/mL). 20 mL of ethyl acetate was added. The microwave was launched with

126

the program described in Table 1.

127

The organic layer was filtered through anhydrous sodium sulfate, evaporated in a rotary

128

evaporator, and subsequently placed under a gentle stream of nitrogen to complete the

129

evaporation.

130

For infant formula. The sample was homogenized. For liquid formula the sample was

131

stirred for several minutes; while for powdered formula, the sample was prepared using

132

water (the percentage of milk reconstitution used was as per the recommendations on

133

the package, i.e. approximately 13.5%.) and then homogenized. 5 mL of infant formula

134

was spiked with 50 µL of the stock solution of 3-MCPD d5 ester (40 µg/mL) and 50 µL of

135

the standard solution of glycidyl d5 ester (50 µg/mL), then 20 mL of ethyl acetate was

136

added. The microwave was launched with the program described in Table 1, and the

137

sample was centrifuged at 4300 rpm for 5 minutes. The organic layer was filtered

calculated using the following formula: x =104/y



138

through anhydrous sodium sulfate, evaporated in a rotary evaporator, then placed under

139

a gentle stream of nitrogen to complete the evaporation.

140 141

Procedure for esters cleavage and matrix clean-up.9 600 µL of diethyl ether was

142

added to the extract and the calibration solutions in order to completely dissolve the

143

sample material. The mixture was shaken shortly and stored for 30 min in a freezer to

144

cool down to -22 to -25°C. If the sample was precipitated, 350 µL of methanolic sodium

145

hydroxide solution was added and the mixture was shaken briefly before being stored for.

146

16 to 20 hours in a freezer to cool down to -22 to -25°C. The reaction was stopped by

147

the addition of 600 µL of sodium bromide acid solution and the mixture was shaken

148

briefly. The solution was placed under a gentle stream of nitrogen to approximatively

149

100 µL, then 600 µL of n-heptane was added to the mixture and shaken vigorously. The

150

organic layer was then discarded. This step was repeated with 600 µL of n-heptane. The

151

aqueous layer was extracted three times with 600 µL of a mixture of diethyl ether/ethyl

152

acetate (60/40) and the organic layers were combined in a new vial and 50 µL of 1,2-

153

hexanediol (0.5 µg/mL) was added. This solution was derivatized as described below.

154 155

Derivatization Procedure.9 100 µL of phenylboronic acid (20 mg/mL) was added to the

156

organic layer ; the mixture was shaken vigorously and then allowed to stand for 1 hour

157

before being evaporated under a stream of nitrogen. The derivatives were extracted by

158

shaking with 500 µL of iso-octane which were transferred in a microinsert and analyzed

159

by GC-MS.

160


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Method Validation. The samples used for the validation (extra virgin sunflower oil,

162

potato crisps, chips cooked in a restaurant, powder infant formula) were purchased from

163

stores and selected randomly. Some aliquots of interlaboratory test (FAPAS Test 2642

164

and some aliquots of the AOCS Collaborative study (Cd 30-15 coupled with Cd 29a-13)

165

based on the work of Ermacora and Hrnčiřík30) were also used for oils and oil-based

166

emulsions such as margarines and spreads.

167

Quantitation limits were set according to the European Recommendation 2014/661.26

168

The recoveries for esters were determined for each sample using the ratio of the peaks

169

area responses for 1-2-hexanediol and internal standard. Another compound (1-2-

170

hexandiol) was used for the determination of recoveries, specifically the recovery of

171

MCPD esters and the recovery of glycidyl esters. They were set according to the

172

recommendation of COFRAC guidelines.31

173

For testing the method, samples with very low concentrations of esters were used. The

174

samples were analyzed ten times and the average concentration allowed us to set a

175

value of "blank sample". These “blank samples” were spiked at three levels: 0.1, 0.5 and

176

10 mg/kg for oils; 0.02, 1 and 5 mg/kg for chips and crisps and 0.0025, 0.05 and 0.2

177

mg/L for infant formula. These samples were then analyzed using the described method

178

at least in duplicate per day, and five replicates of these series were performed by two

179

different operators for five days. Recoveries, repeatability and reproducibility (SD), were

180

determined from this data series.

181

The linearity of the method was checked by the analysis of nine standard solutions in the

182

same conditions as described above. The LOQ was verified by the analysis of the “blank”

183

sample spiked at the respective levels (0.1 mg/kg for oils; 0.02 mg/kg for chips and

184

crisps and 0.0025 mg/L for infant formula).



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This method was also validated for mayonnaise and butter products using an aliquot of a

186

certified reference material by AOCS.

187 188

GC-MS Analysis. For this study, a GC-MS Shimadzu QP 2010 Plus and a split/splitless

189

injection system were used. The separation was performed using a HP 5 MS capillary

190

column (Agilent - 5% phenyl, 95% dimethylpolysiloxane – 30 m x 0.25 m x 0.25 µm film

191

thickness). The injector temperature was kept at 250°C and the carrier gas was Helium

192

5.0 with a constant flow of 1.7 mL/min. The transfer line was at 250°C, the ion source at

193

230°C and the quadrupole at 150°C. The GC column oven was programmed from an

194

initial temperature of 80°C held for 1 min, increased at a rate of 10°C/min up to 200°C,

195

and then ramped up again at 15°C/min up to 250°C, wich was held for 15 min. The total

196

run time was 31.33 mins. The mass selective detector was used for selected ion

197

monitoring, focusing on the following ions :

198

(m/z) ratio of 147 (target) m/z = 196, 198 (qualifiers) for 3-MCPD derivatives

199

(m/z) ratio of 150 and 201 (target) m/z = 201, 203 (qualifiers) for 3-MCPD d5 derivatives.

200

(m/z) ratio of 147 (target) m/z = 240, 242 (qualifiers) for 3-MBPD derivatives.

201

(m/z) ratio of 150 (target) m/z = 245, 247 (qualifiers) for 3-MBPD d5 derivatives.

202

(m/z) ratio of 196 (target) m/z = 196, 198 (qualifiers) for 2-MCPD derivatives.

203

(m/z) ratio of 147 (target) m/z = 204 (qualifiers) for 1-2-hexanediol derivatives.

204

The dwell time for each m/z was 50.

205 206

Quantitation. The concentrations of esters were calculated as ratio of the peak area

207

responses for 3-MCPD (m/z 147) and the internal standard (3-MCPD d5, m/z 150) for

208

calibration standards as well as blank and spiked samples; as ratio of the peak area


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responses for 2-MCPD (m/z 196) and the internal standard (3-MCPD d5, m/z 201) for

210

calibration standards as well as blank and spiked samples; as ratio of the peak area

211

responses for 3-MBPD (m/z 147) and the internal standard (3-MBPD d5, m/z 150) for

212

calibration standards as well as blank and spiked samples. The respective

213

concentrations were determined from a calibration graph constructed by plotting the

214

peak area ratios for the calibration standards against the amount of esters.

215

The recovery of extraction was determined as follows: = 100 ∗

216 217

⁄ ⁄ ∗ ⁄ . ⁄ .

Where : •

⁄ corresponds to the ratio of the peak area responses for the

218

internal standard (3-MCPD d5, m/z 150 for the 3-MCPD recovery; 3-MCPD d5,

219

m/z 201 for the 2-MCPD recovery and 3-MBPD d5, m/z 150 for the 3-MBPD

220

recovery) and 1-2-hexanediol (m/z 147) for the sample.

221

•

⁄ . corresponds to the mean of the ratio of the peak area

222

responses for the internal standard (3-MCPD d5, m/z 150 for the 3-MCPD

223

recovery; 3-MCPD d5, m/z 201 for the 2-MCPD recovery and 3-MBPD d5, m/z

224

150 for the 3-MBPD recovery) and 1-2-hexanediol (m/z 147) for the calibration

225

standards.

226

•

⁄ corresponds to the ratio of the concentration of 1-2-hexanediol

227

and the concentration of the internal standard (3-MCPD ester d5 for the MCPD

228

recovery and glycidyl ester d5 for the 3-MBPD recovery) for the sample.

229 230

•

⁄ . corresponds to the mean of the ratio of the concentration of 12-hexanediol and the concentration of the internal standard (3-MCPD ester d5 for



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231

the MCPD recovery and glycidyl ester d5 for the 3-MBPD recovery) for the

232

calibration standards.

233 234

RESULTS AND DISCUSSION

235

The reaction scheme was shown in Figure 1.

236 237

Microwave extraction. A microwave was used for the extraction of fat. There were

238

several advantages of using a microwave reactor rather than a conventional oven for

239

extractions which include: the significant time savings (only 10 minutes), the automatic

240

sample processing, less solvent amounts were required, closed vessel conditions

241

prevent evaporation of the solvent, and the accuracy of the temperature sensor allowed

242

for improved reproducibility. Furthermore, the extraction of fat in foodstuffs using

243

microwave yielded good results compared to conventional methods like the Röse-

244

Gottlieb method.

245

Ethyl acetate was incorporated rather than tert-butyl methyl ether, although it is used for

246

determination of concentrations of esters in margarine and butter products.7,9 The best

247

extraction yields were achieved with ethyl acetate, particularly for infant formula, as an

248

emulsification occurs with tert-butyl methyl ether (yield #$% − 60% ∗ #

%$290

•

and ̅ + 2 ∗ ! < #$% + 60% ∗ #

%$291

The LOQ was checked with an EMA of 60%; this value was defined by convention in NF

292

T90-210. It can be explained as follows: according Horwitz, the maximum coefficient of

293

variation (CV) is defined as CVmax Horwitz = 22%. Therefore, the CVmax of the method can

294

be defined as CVmax of method = CVHorrat * CVmax Horwitz = 2*22% = 44%, that is to say that

295

EMAmax (%) = 2* CVmax of method = 88%. So, the EMA of 60% was acceptable.

296

The detection limit was estimated to: LOQ / 3. The different limits determined in the

297

context of this study are shown in Table 2.

298 299

Accuracy. The method of validation was performed according to standard NF-T90-

300

210.32 In this standard, it is necessary to verify that the results, which are obtained on a

301

material associated to a reference value in intermediate precision conditions, are


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302

acceptable compared to a maximum acceptable deviation (EMA). They are previously

303

defined and fixed by a regulatory, or prescriptive requirement, or the laboratory.

304

This standard provides a study of accuracy of the method on at least three levels of

305

scope. This study leads to the determination of the performance parameters

306

(repeatability and reproducibility), detection and quantitation limits, and recovery. To

307

define the performance of a non-standard method, the accuracy study is completed by a

308

study of linearity and accuracy.

309

This standard was originally written for the field of water. However the principles of

310

validation and experimental designs are identical to those published in other standards

311

or validation guides from other fields (wines, contact materials, pesticide, etc.).

312

A full validation report was performed for each matrix.

313

The accuracy of the method was checked for various foodstuffs like oils, chips, crisps

314

and infant formulas; the accuracy of which was investigated on three different levels.

315

This study led to the determination of different parameters: repeatability, reproducibility,

316

recoveries, LOQ and LOD. It was performed:

317

• on samples with very low and known analyte concentrations, which are spiked at the

318

beginning of the analysis at concentration levels studied.

319

• on aliquots of interlaboratory tests (FAPAS Test 2642 and some aliquots of the AOCS

320

Collaborative study (Cd 30-15 coupled with Cd 29a-13) based on the work of Ermacora

321

and Hrnčiřík30).

322

Target values were matched either to the theoretical values of spiking, or to the value

323

provided by the results of interlaboratory tests. For each level, the analysis was

324

performed five times (reproducibility conditions) in duplicate (repeatability requirement)

325

with different operators. For each duplicate, a new calibration curve was drawn.



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326

NF T90-21032 proposes to verify the accuracy of the method around a reference value

327

relative to the maximum acceptable deviation (EMA) which describes the performance of

328

the method. The EMA (maximum deviation allowed) which was set by the laboratory,

329

was 60% for the first level and 30% for the others. These values were chosen according

330

to NF T90-21022 for LOQ and according to Horwitz (EMA% = 2* CVHorwitz = 2 * 2 * C-0.15

331

where C is the concentration found or added, expressed as a mass fraction) for other

332

levels. It is recommended to graph the accuracy of the method by level according to the

333

tolerance interval of the relative bias, with respect to the maximum acceptable deviation

334

(EMA) in percentage. This is called the " accuracy profile of the method", and the results

335

are shown in Figure 3-5.

336

Following to the recommendation of COFRAC guidelines21, it was verified that the

337

recovery of extraction was between 30 and 120% with a maximum CV of 20%.

338

The accuracy of the method was also verified through the analyses of several

339

interlaboratory tests (13 assays).

340 341

Quality control. The performance of the method was monitored throughout the

342

validation of the method with different means. Analysis of a blank, specifically extra

343

virgin sunflower oil, and a quality control sample was conducted at each injection

344

sequence. The sample of quality control was an old sample of an interlaboratory test

345

(FAPAS Test 2642). This is an oil containing the three esters. This data was combined

346

on a Shewhart control chart (see Figure 6-8).

347

The target value and limits of monitoring and control correspond to the results provided

348

by the interlaboratory test (FAPAS Test 2642). Routinely, these charts allow us to detect


Page 17 of 45


349

any drift due to the measuring apparatus, extraction, or the derivatization of compounds.

350

In addition, the extraction yields were checked for each analysis.32

351 352

In conclusion, due to the simplicity, rapidity, and economy, the presented method can be

353

used as a method for routine analysis of various foodstuffs. The method has the

354

advantage of minimal sample preparation, low LOQ, and good repeatability and

355

reproducibility for the analysis of these esters.

356 357 358

ABBREVIATIONS AND NOMENCLATURE

359

3-MCPD, 3-monochloropropane-1,2-diol; 2-MCPD, 2-monochloropropane-1,3-diol; CV,

360

coefficient of variation; GC-MS, gas chromatography-mass spectrometry; EU, European

361

Commission; LOQ, limit of quantitation: LOD, limit of detection; TRC, Toronto Research

362

Chemicals; PBA, phenylboronic acid; COFRAC, French Accreditation Comittee; AOCS,

363

American Oil Chemists' Society; EMA, maximum deviation allowed

364 365 366

ACKNOWLEDGEMENT

367

/

368 369 370

FUNDING SOURCES

371

/



372 373


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1. C.G. Hamlet, P.A. Sadd, C. Crews, J. Velisek, D.E. Baxter, Occurrence of 3-

376

chloro-propane-1,2-diol (3-MCPD) and related compounds in foods: a review,

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Food Addit. Contam., 2002, 19, 619-631

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2. I. Baer, B. de la Calle, P. Taylor, 3-MCPD in food other than soy sauce or

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hydrolysed vegetable protein (HVP), Anal. Bioanal. Chem., 2010, 396, 443-56

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3. European Commission. Setting maximum levels for certain contaminants in

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foodstuffs.: Commission Regulation (EU) No 1881/2006. Official Journal of the

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European Union. 2006, L364, 5

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Regulation (EU) No 333/2007 laying down the methods of sampling and analysis

385

for the official control of the levels of lead, cadmium, mercury, inorganic tin, 3-

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MCPD and benzo(a)pyrene in foodstuffs. 2011, L215, 9

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chlorohydrins and their esters as products of the hydrolysis of tripalmitin,

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tristearin and triolein with hydrochloric acid. Z. Lebens. Unters. Forsch. Janvier

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1980, Vol. 171, N°1

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6. Cerbulis J., Parks O.W., Liu R.H., Piotrowski E.G., Farrell H.M. Occurrence of

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diesters of 3-chloro-1,2-propanediol in the neutral lipid fraction of goats' milk. J.

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Agric. Food Chem. 1984, 32, 474-476.

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7. MacMahon S., Begley T. H., Diachenko G. W., Occurrence of 3-MCPD and

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glycidyl esters in edible oils in the United States, Food Addit. Contam. 2013, 30,

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2081-2092.



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8. ILSI Europe Report Series, Meeting organised by the ILSI Europe Process-

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related Compounds and Natural Toxins Task Force and Risk Assessment of

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Chemicals in Food Task Force in association with the European Commission

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(EC) and the European Food Safety Authority (EFSA), Brussels, Belgium, Feb.

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2009.

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9. Kuhlmann J. Determination of bound 2,3-epoxy-1-propanol(glycidol) and bound

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monochloropropanediol (MCPD) in refined oils. Eur. J. Lipid Sci.Tech. 2011, 113,

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335-344.

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10. EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain),

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Statement on a request from the European Commission related to 3-MCPD

407

esters, http://www.efsa.europa.eu/en/efsajournal/pub/1048, Question number :

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EFSA-Q-2008-258, Adopted: 28 March 2008, doi:10.2903/j.efsa.2008.1048

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11. Buhrke T., Weisshaar R., Lampen A., Absorption and metabolism of the food

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contaminant 3-chloro-1,2-propanediol (3-MCPD) and its fatty acid esters by

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human intestinal Caco-2 cells. Arch. Toxicol. 2011, 85, 1201-1208

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12. Barocelli E., Corradi A., Mutti A., Petronini P.G., Scientific Report submitted to

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EFSA "Comparison between 3-MCPD and its palmitic esters in a 90-day

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toxicological study, 2011, http://www.efsa.europa.eu/en/supporting/pub/187e.htm

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13. K.E. Appel, K. Abraham, E. Berger-Preiss, T. Hansen, E. Apel, S. Schuchard, C.

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Vogt, N. Bakhya, O. Creutzenberg, A.Lampen: Relative oral bioavailability of

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glycidol from glycidyl fatty acid esters in rats, Arch. Toxicol. 2013, 87(9), 1649-59

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14. Buhrke T, Frenzel F, Kuhlmann J, Lampen A., 2-Chloro-1,3-propanediol (2-

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MCPD) and its fatty acid esters: cytotoxicity, etabolism, and transport by human

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intestinal Caco-2 cells, Arch Toxicol., 2015, 89(12), 2243-51


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15. Monographs on the Evaluation of Carcinogenic Risks to Humans. Volume 77

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Some Industrial Chemicals. IARC (International Agency for Research on

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Cancer), 2000. Summary of Data Reported and Evaluation, Lyon, France, 469-

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486, http://monographs.iarc.fr/ENG/Monographs/vol77/

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16. Some Chemicals Present in Industrial and Consumer Products, Food and

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Drinking–Water. IARC (International Agency for Research on Cancer), 2012,

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101, 349-374. http://monographs.iarc.fr/ENG/Monographs/vol101/

428

17. Glycidyl Fatty Acid Esters in Edible Oils. AOCS/JOCS Official Method Cd 28-10,

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https://aocs.personifycloud.com/PersonifyEbusiness/Store/ProductDetails.aspx?

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productId=111541

431

18. AOCS : Official Method Cd 29a-13 Approuved 2013. 2-and 3-MCPD Fatty Acid

432

Esters and Glycidol Fatty Acid Esters in Edible Oils and Fats by Acid

433

Transesterification, Official Methods and Recommended Practices of the AOCS,

434

2013a

435

19. AOCS : Official Method Cd 29b-13 Approuved 2013. Determination of Bound

436

Monochloropropanediol-(MCPD-) and Bound 2,3-epoxy-1-propanol (glycidol-) by

437

Gas Chromatography/Mass Spectrometry (GC/MS), Official Methods and

438

Recommended Practices of the AOCS, 2013b

439

20. AOCS : Official Method Cd 29c-13 Approuved 2013, Fatty-acid-bound 3-

440

chloropropane-1,2-diol

(3-MCPD)

and

2,3-epoxi-propane-1-ol

(glycidol),

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Determination in Oils and Fats by GC/MS (Differential Measurement), Official

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Methods and Recommended Practices of the AOCS, 2013c

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21. Wenzl T., Samaras V., Giri A., Buttinger G., Karasek L., Zelinkova Z.

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Development and validation of analytical methods for the analysis of 3-MCPD



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(both in free and ester form) and glycidyl esters in various food matrices and

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performance of an ad-hoc survey on specific food groups in support to a scientific

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opinion on comprehensive risk assessment on the presence of 3-MCPD and

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glycidyl esters in food. EFSA supporting publication 2015 : EN-779.

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http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documen

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ts/779e.pdf

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22. BfR method validation study for method 22: H. Fry, C. Schödel, A. These , A.

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Preiß-Weigert: Collaborative Study for the Determination of 3-MCPD- and 2-

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MCPD- Fatty Acid Esters in Fat Containing Foods, Federal Institute for Risk

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Assessment (BfR), 2013

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23. H. Karl, S. Merkle, J. Kuhlmann, J. Fritsche, Development of analytical methods

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for the determination of free and ester bound 2-, 3-MCPD, and esterified glycidol

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in fishery products, Eur. J. Lipid Sci. Technol. 2015, 118, 406-417

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24. M. Küsters et al.; Simultaneous Determination and Differentiation of Glycidyl

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Estersand 3-Monochloropropane-1,2-diol (MCPD) Esters in Different Foodstuffs

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by GC-MS, J. Agric. Food Chem. 2011, 59, 6263–6270

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25. AOCS : Official Method. Analysis of 2-and 3-MCPD Fatty Acid Esters and

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Glycidol Fatty Acid Esters in Oils-Based Emulsions, AOCS Official Method

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CD30-15 : in project.

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26. Commission recommendation on the monitoring of 2- et 3-monochloro-propane-

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1,2-diol (2- et 3-MCPD), 2- and 3-MCPD esters and glycidyl esters in foodstuffs.

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European Commission : Commission Recommendation (EU) No 661/2014. Off.

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J. Eur. Communities, 2014, L271, 93-95


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27. Sandroni V., Smith C.M.M., Donovan A. Microwave digestion of sediment, soils

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28. Kamankesh M., Mohammadi A., Hosseini H., Modarres Tehrani Z., Rapid

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determination of polycyclic aromatic hydrocarbons in grilled meat using

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coupled to gas chromatography-mass spectrometry. Meat Sci., 2015 May, 103 :

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61-7.

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29. García-Ayuso LE, Velasco J, Dobarganes MC, Luque De Castro MD.,

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Accelerated extraction of the fat content in cheese using a focused microwave-

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assisted soxhlet device, J Agric Food Chem., 1999 Jun, 47(6) : 2308-15.

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30. Ermacora A., Hrnčiřík K. Development of an analytical method for the

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Food Addit. Contam., 2014, 31, 985-994.

482 483 484 485

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486

CAPTIONS OF FIGURES AND TABLES

487

Table 1. Optimal condition for microwave.

488

Table 2. Conclusion about LOQ and LOD

489

Figure 1A. Procedure for esters cleavage

490

Figure 1B. Derivatization procedure

491

Figure 2A. Blank samples : extra virgin oil for 3-MCPD esters

492

Figure 2B. Blank samples : extra virgin oil for 2-MCPD esters

493

Figure 2C. Blank samples : extra virgin oil for 2-MCPD esters

494

Figure 3A. Accuracy profile for oils for 3-MCPD esters

495

Figure 3B. Accuracy profile for oils for 2-MCPD esters

496

Figure 3C. Accuracy profile for oils for glycidyl esters

497

Figure 4A. Accuracy profile for crisps and chips for 3-MCPD esters

498

Figure 4B. Accuracy profile for crisps and chips for 2-MCPD esters

499

Figure 4C. Accuracy profile for crisps and chips for glycidyl esters

500

Figure 5A. Accuracy profile for infant formula for 3-MCPD esters

501

Figure 5B. Accuracy profile for infant formula for 2-MCPD esters

502

Figure 5C. Accuracy profile for infant formula for glycidyl esters

503

Figure 6. Control charts on 3-MCPD esters (LCS and LCI : Control Limits, LSS and LSI:

504

monitoring limits)

505

Figure 7. Control charts on 2-MCPD esters (LCS and LCI : Control Limits, LSS and LSI:

506

monitoring limits)

507

Figure 8. Control charts on glycidyl esters (LCS and LCI : Control Limits, LSS and LSI:

508

monitoring limits)

509


Page 24 of 45

Page 25 of 45


510

ASSOCIATED CONTENT

511

Table S1. Comparison of the extraction of fat between the Röse Gottlieb method and

512

the microwave method.

513

Table S2. Microwave extraction : comparison between tert-butyl methyl ether and ethyl

514

acetate and between stirrings rods oval-shaped and rectangular stirring rods.

515

Table S3. Results of linearity study for 3-MCPD esters

516

Table S4. Results of linearity study for 2-MCPD esters

517

Table S5. Results of linearity study for glycidyl esters

518

Table S6. Results of checking of LOQ for oils

519

Table S7. Results of checking of LOQ for crisps and chips

520

Table S8. Results of checking of LOQ for infant formula

521

Table S9. Results of accuracy for oils

522

Table S10. Results of accuracy for crisps and chips

523

Table S11. Results of accuracy for infant formula

524

Table S12. Results of recovery for oils

525

Table S13. Results of recovery for crisps and chips

526

Table S14. Results of recovery for infant formula

527

Table S15. Results of inter laboratory tests

528 529

CONFLICT OF INTEREST

530

The

531

TABLES AND FIGURES

authors

declare

no

competing

532 533

Table 1. Optimal condition for microwave.


financial

interest.


Step

Temp. (°C)

Fast heating

60

Hold time Cooling

Time (hh:mm:ss)

Stirring speed (rpm)

00:10:00

600

55

534 535 536


Page 26 of 45

Page 27 of 45

537


Table 2. Conclusion about LOQ and LOD Oils 3-MCPD esters

2-MCPD esters

Glycidyl esters

LOQ (mg/kg)

0.1

0.1

0.1

LOD (mg/kg)

0.03

0.03

0.03

Crips and chips LOQ (mg/kg)

0.02

0.02

0.02

LOD (mg/kg)

0.007

0.007

0.007

Infant formula LOQ (mg/L)

0.0025

0.0025

0.0025

LOD (mg/L)

0.0008

0.0008

0.0008

538 539 540 541 542



543

Figure 1A. Procedure for esters cleavage For 3-MCPD esters :

For 2-MCPD esters :

For glycidyl esters :

544


Page 28 of 45

Page 29 of 45

545


Figure 1B. Derivatization procedure For 3-MCPD esters :

For 2-MCPD esters :

For glycidyl esters :

546



547

Figure 2A. Blank samples : extra virgin oil for 3-MCPD esters

m/z 147 : 3-MCPD derivative

m/z 150 : 3-MCPD d5 derivative 548


Page 30 of 45

Page 31 of 45

549


Figure 2B. Blank samples : extra virgin oil for 2-MCPD esters

m/z 196 et 198 : 2-MCPD derivative

m/z 201 and 203 : 3-MCPD d5 derivative 550 551



552

Figure 2C. Blank samples : extra virgin oil for 2-MCPD esters

m/z 197 : Glycidyl derivative

m/z 150 : Glycidyl d5 derivative 553


Page 32 of 45

Page 33 of 45


554 555

Figure 3A. Accuracy profile for oils for 3-MCPD esters

556 557



558

Figure 3B. Accuracy profile for oils for 2-MCPD esters

559 560


Page 34 of 45

Page 35 of 45

561


Figure 3C. Accuracy profile for oils for glycidyl esters

562 563



564

Figure 4A. Accuracy profile for crisps and chips for 3-MCPD esters

565 566


Page 36 of 45

Page 37 of 45

567


Figure 4B. Accuracy profile for crisps and chips for 2-MCPD esters

568 569



570

Figure 4C. Accuracy profile for crisps and chips for glycidyl esters

571 572


Page 38 of 45

Page 39 of 45

573


Figure 5A. Accuracy profile for infant formula for 3-MCPD esters

574 575



576

Figure 5B. Accuracy profile for infant formula for 2-MCPD esters

577 578


Page 40 of 45

Page 41 of 45

579


Figure 5C. Accuracy profile for infant formula for glycidyl esters

580 581 582



583

Figure 6. Control charts on 3-MCPD esters (LCS and LCI : Control Limits, LSS and LSI: monitoring limits)

584

585 586 587


Page 42 of 45

Page 43 of 45

588


Figure 7. Control charts on 2-MCPD esters (LCS and LCI : Control Limits, LSS and LSI: monitoring limits)

589

590 591



592

Figure 8. Control charts on glycidyl esters (LCS and LCI : Control Limits, LSS and LSI: monitoring limits)

593

594 595 596


Page 44 of 45

Page 45 of 45


TOC Graphic


Determination of 3-Monochloropropane-1, 2-diol and 2

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