Applicability of Gas Chromatography (GC) Coupled to Triple

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Applicability of gas chromatography (GC) coupled to triple quadrupole (QqQ) tandem mass spectrometry (MS/MS) for polybrominated diphenyl ether (PBDE) and emerging brominated flame retardants (BFR) determinations in functional foods enriched in omega-3 Angel Garcia-Bermejo, Susana Mohr, Laura Herrero, Maria Jose Gonzalez, and Belén Gomara J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03118 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 12, 2016

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Page 1 of 35

Journal of Agricultural and Food Chemistry

1

Applicability of gas chromatography (GC) coupled to triple quadrupole (QqQ)

2

tandem mass spectrometry (MS/MS) for polybrominated diphenyl ether (PBDE)

3

and emerging brominated flame retardants (BFR) determinations in functional

4

foods enriched in omega-3.

5

6

Ángel García-Bermejo1, Susana Mohr1,2, Laura Herrero1, María-José González1, Belén

7

Gómara1*

8 9

1

Department of Instrumental Analysis and Environmental Chemistry, Institute of

10

General Organic Chemistry IQOG-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain

11

2

12

(UFSM), Av. Roraima 1000, Prédio 42, Sala 3135, 97105-900, Santa María, RS, Brazil

Department of Food Science and Technology, Federal University of Santa María

13 14

*

15

address [email protected], telephone number +34915622900

Author to whom inquiries about the paper should be addressed: Belén Gómara, e-mail

16

1 ACS Paragon Plus Environment


17

Abstract

18

This paper reports on optimization, characterization, and applicability of gas

19

chromatography coupled to triple quadrupole tandem mass spectrometry (GC-

20

QqQ(MS/MS)) for the determination of 14 polybrominated diphenylethers (PBDEs) and

21

two emerging brominated flame retardants, 1,2-bis(2,4,6-tribromophenoxy)ethane

22

(BTBPE) and decabromodiphenylethane (DBDPE), in functional food samples. The

23

method showed satisfactory precision and linearity with instrumental limits of detection

24

(iLODs) ranging from 0.12 to 7.1 pg, for tri- to octa-BDEs and BTBPE, and equal to 51

25

and 20 pg for BDE-209 and DBDPE, respectively. The highest ΣBFR concentrations

26

were found in fish oil supplements (924 pg/g fresh weight, f.w.), followed by biscuits

27

(90 pg/g f.w.), vegetable oil supplements (46 pg/g f.w.), chicken eggs (45 pg/g f.w.),

28

cow's milk (7.7 pg/g f.w.) and soy products (1.6 pg/g f.w.). BDEs 47, 99, and DBDPE

29

were the most abundant compounds. Foodstuffs enriched with omega-3 presented

30

similar or even lower concentrations than conventional foods commercialized in Spain

31

since 2000.

32

33

Keywords: Gas chromatography; triple quadrupole mass spectrometry; polybrominated

34

diphenyl ethers; emerging brominated flame retardants; functional foods; omega-3.

35


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36

INTRODUCTION

37

Brominated flame retardants (BFRs), such as polybrominated diphenyl ethers (PBDEs),

38

are synthetic reactive or additive chemicals that have been widely added in

39

consumer/commercial products, i.e. computers, mobile phones, electrical kitchen

40

appliances, textiles, building materials and many plastic products, to reduce their

41

flammability and thereby prevent or retard the spread of fires. They have been in use

42

since the early 1970s 1. PBDEs are a class of BFRs that have been used more

43

extensively in a wide range of consumer products, and therefore have been produced in

44

large quantities 2,3. There have been three major PBDE commercial formulations in the

45

global market: Penta-, Octa- and Deca-BDEs. Because of PBDEs are mixed into

46

polymers and not chemically bound to the plastics or textiles, they might separate or

47

leach from the consumer products into the environment 4. Consequently, these

48

substances have over time contaminated the environment and the food chain, since

49

consumer goods are discarded at the end of their life. Most BFRs, including the PBDEs,

50

are persistent, bioaccumulative, and toxic or neurotoxic and can be potentially

51

dangerous for human and environmental health as described in toxicological studies 5,6.

52

Because of that, currently, those PBDEs present in the Penta- and Octa-BDE

53

commercial formulations have been listed under the United Nations Environment

54

Programme`s Stockholm Convention on Persistent Organic Pollutants (POPs) and the

55

Deca-BDE commercial product is currently being considered for listing as a POP under

56

the same convention 7. On the other hand, since the European Union (EU) took actions

57

on the use and applications of PBDEs (the use of Penta- and Octa-BDE technical

58

mixtures were banned in 2003 8 and Deca-BDE technical mixture in 2008 9) alternative

59

BFRs,

60

decabromodiphenylethane (DBDPE), are being used as replacements of Octa- and

such

as

1,2-bis(2,4,6-tribromophenoxy)ethane


(BTBPE)

and


61

Deca-BDE formulations, respectively, becoming “emerging” BFRs. BTBPE is

62

marketed for use in acrylonitrile butadiene styrene (ABS), high-impact polystyrene

63

(HIP), thermoplastics, thermoset resins, polycarbonates, and coatings 10. DBDPE is used

64

as an additive for different polymeric materials such as HIP, polypropylene, and ABS

65

and in textiles, such as polyester and cotton 10. These compounds have similar structures

66

to PBDEs and they are expected to have similar physical-chemical properties. It is well-

67

known that dietary intake is considered an important exposure pathway to PBDEs,

68

along with ingestion of indoor dust and inhalation of indoor air, and the similar

69

physicochemical properties of the emerging BFRs, suggest that they will follow a

70

similar exposure pattern to PBDEs

71

PBDEs and emerging BFRs in the human food chain 12-27.

72

On the other hand, nowadays, in the food industry, the use of additives to result in

73

functional foods such as food enriched with omega-3 polyunsaturated fatty acids

74

(PUFAs) is very common. This enrichment generally occurs through the addition of fish

75

oils to the food product since they are rich in omega-3 PUFAs, EPA (eicosa-pentaenoic-

76

acid) and DHA (docosa-hexaenoic-acid). However, it is well known that, fatty fishes,

77

with a high content of omega-3 PUFAs, may also contain relatively high concentrations

78

of POPs

79

general foodstuffs but, even more, in aforementioned enriched food products, in order to

80

determine if their composition could represent a human health risk. With regard to this,

81

it is important to highlight that, to date, few investigations on PBDE levels in foodstuffs

82

have been reported for Spain

83

substitutes for Octa- and Deca-BDE formulations, and only one deals with omega-3

84

dietary supplements. Therefore, this is the first study on PBDEs and emerging BFRs in

85

commercial foodstuffs enriched with omega-3 PUFAs in Spain.

11

. In fact, some studies have already reported

28,29

. So, it is very interesting to evaluate the presence of BFRs, not only in

16-20,26

, neither includes the two main emerging BFR


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86

Regarding instrumental determination of BFRs, while most studies use gas

87

chromatography coupled to single quadrupole mass spectrometry (GC-Q(MS)) in both

88

electron ionization (EI) and electron capture negative ionization (ECNI) for PBDE and

89

emerging BFR determinations, and focus on the analysis of environmental and human

90

matrices 30-36, other works are reported using GC coupled to triple quadrupole MS in its

91

tandem operation mode (GC-QqQ(MS/MS)) for the analysis of these compounds in

92

environmental and biological samples

93

literature dealing with the determination of BFRs in foodstuffs by GC-QqQ(MS/MS)

94

12,21,42

95

determinations.

96

For all these reasons, the aim of this work was to develop an analytical method for

97

PBDE and emerging BFR determination by GC-QqQ(MS/MS) and to study its

98

applicability for the identification and quantification of 14 PBDE congeners (from tri-

99

to deca- substituted) and two emerging brominated flame retardants (1,2-bis(2,4,6-

100

tribromophenoxy)ethane (BTBPE) and decabromodiphenylethane (DBDPE)) in

101

different commercially available foodstuffs enriched in omega-3 PUFAs. Additionally,

102

results will be compared to those found by other authors in similar surveys in order to

103

further assess the applicability of the technique for this kind of analysis and to evaluate

104

the benefits and risk of functional foods enriched with omega-3 PUFAs.

105

MATERIALS AND METHODS

106

Reagents and standards.

107

All solvents used were of Pestipur® quality and were purchased from SDS (Peypin,

108

France), except n-hexane (Merck, Darmstadt, Germany). Sulphuric acid was of pro-

109

analysis quality (Merck). Anhydrous sodium sulphate was obtained from J. T. Baker

37-41

. However, few works are found in the

, despite previous works evidencing that it is a suitable technique for this kind of



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110

(Deventer, The Netherlands) and silica gel 60 from Merck. Solid phase extraction (SPE)

111

cartridges of SupelcleanTM Envi-CarbTM (graphitized carbon pack, 250 mg, 3 mL tubes;

112

Supelco, Palo Alto, USA) were used for final fractionation and clean-up.

113

The congeners selected for this study were chosen due to their abundance in the

114

technical mixtures and occurrence in the environment and in food. They are relevant for

115

dietary exposure and they were considered by the European Food Safety Authority

116

(EFSA) Panel on Contaminants in the Food Chain to be of primary interest (CONTAM

117

Panel) 1. Thus, BDEs 17, 28, 47, 66, 99, 100, 153, 154, 183, 184, 191, 196, 197, and

118

209, and two alternative BFRs used as replacement of Octa- and Deca-BDE

119

formulations, BTBPE and DBDPE, respectively, were chosen.

120

All the standards solutions were purchased from Wellington Laboratories Inc. (Guelph,

121

Ontario, Canada) and Cambridge Isotope Laboratories (Andover, MA, USA). A

122

solution of 20 pg/µL of labeled

123

spiking/surrogate standard, and a solution of 20 pg/µL of labeled

124

138 was used as an injection standard for recoveries calculation.

125

Precision was evaluated using three different concentration levels (low, medium, and

126

high), i.e. 10, 50, and 100 pg/µL for tri- to hepta-BDEs and BTBPE; 30, 150, and 300

127

pg/µL for octa-BDEs; and 100, 500, and 1000 pg/µL for BDE-209 and DBDPE. The

128

repeatability (intraday precision) was calculated as the relative standard deviation (RSD,

129

%) of the areas and ion transition ratios corresponding to four consecutive injections

130

and the intermediate precision (interday precision) was expressed as the RSD (%) of the

131

areas and ion transition ratios of four injections carried out in different days along two

132

weeks. It should be noted that, in the case of QqQ analyzers, the ion transition ratio is

133

the ratio between the intensities of the two transitions selected in the optimization

13

C12-BDEs 47, 99, 100, and 153 was used as a


13

C12-BDEs 77 and

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134

process for each compound. Linearity was evaluated using a set of five calibration

135

standard solutions of the 14 native PBDEs containing the corresponding

136

compounds plus BTBPE and DBDPE, in nonane. The concentrations of native

137

compounds ranged from 1 to 500 pg/µL, except for BDE-209 and DBDPE which

138

ranged from 5 to 2500 pg/µL and from 10 to 5000 pg/µL, respectively. The

139

labeled compound concentrations were 20 pg/µL. Since for GC-QqQ(MS/MS)

140

instruments it is not appropriate the use of the classical signal to noise ratio (S/N)

141

approach for calculating instrumental limits of detection (iLODs) as the amount of

142

analyte which produces a S/N equal to three, because to the very low noise generated by

143

the instrument

144

corresponding to 3 times the standard deviation (SD) of the signal of three replicate

145

injections of a standard solution closed to limit of detection. The relative response of the

146

labeled congeners

147

spiking/surrogate standard before the extraction) against the labeled congeners

148

BDEs 77 and 138 (added as injection standard just before the injection of the final

149

extract in the GC-QqQ(MS/MS) system) in both calibration solutions and sample

150

extracts was used for recovery calculations.

151

Sample collection.

152

Five different types of commercially available functional foods enriched with omega-3

153

PUFAs, i.e. oil dietary supplements of different origin (n=7, six different fish oils and

154

one vegetable (linseed) oil), cow’s milk (n=3), chicken eggs (n=3), soy products (n=3,

155

two soy drinks and one soy lecithin), and biscuits (n=2), were purchased in different

156

supermarkets from Madrid (Spain) between 2010 and 2012, and were analyzed for

157

PBDE and emerging BFR determinations.

13

C12-labeled

13

C12-

43

, in this work, iLODs were calculated as the concentration

13

C12-BDEs 47, 99, 100, and 153 (added to the samples as


13

C12-


Page 8 of 35

158

Sample preparation.

159

The oil dietary supplement samples were extracted and purified according to the method

160

previously described by Guzmán et al., 2005 44, using slight modifications. Briefly, 4 g

161

of oil sample was mixed with 2 mL of n-hexane and spiked with the spiking/surrogate

162

standards. A purification step was performed by using multilayer column filled with

163

neutral silica gel, silica gel activated and modified with sulfuric acid (44% and 22%,

164

w:w), and anhydrous sodium sulfate. A further purification step was carried out on a

165

multilayer column filled with neutral silica gel, silica gel activated and modified with

166

sulfuric acid (44% w:w) and with potassium hydroxide (36% w:w). Finally, the extracts

167

were subjected to a fractionation/clean-up step on Envi-CarbTM SPE cartridges and were

168

eluted with 15 mL n-hexane and 20 mL of n-hexane/toluene (99:1, v:v) for the fraction

169

which contains PBDEs, BTBPE, and DBDPE. The final extracts were transferred to

170

conical bottom injection vials, evaporated to dryness under a gentle nitrogen stream,

171

and reconstituted with the injection standard.

172

For the remaining samples, the treatment described by Bordajandi et al., 2003

173

followed. Briefly, liquid samples (i.e. cow’s milk, chicken eggs, and liquid soy

174

products) were lyophilized, while solid samples (biscuits and soy lecithin) were

175

grounded. Then the fat content of the samples, according to the method published by

176

Smedes 46, was determined. For the extraction, the amount of sample equivalent to 4 g

177

of fat was taken, homogenized with 20 g of anhydrous sodium sulfate and 5 g of

178

activated neutral silica, and extracted by matrix solid phase dispersion (MSPD). The

179

mixed homogenized sample was loaded into a glass column between two layers of

180

granular anhydrous sodium sulfate, and spiking/surrogate standard of PBDEs were

181

added likewise for oil pills samples. The purification and fractionation procedure

182

continues as previously described for oil samples. 8 ACS Paragon Plus Environment

45

, was

Page 9 of 35


183

All the samples were processed in batches, one batch for each type of matrix including a

184

procedural blank too. All samples within a batch were extracted, purified, and

185

concentrated in parallel and all the final extracts were injected just after obtaining them.

186

Instrumental determination by GC-QqQ(MS/MS).

187

Instrumental determination of PBDEs and emerging BFRs (BTBPE and DBDPE) was

188

performed on TRACE GC Ultra gas chromatograph (Thermo Fisher Scientific, Milan,

189

Italy) equipped with a triple quadrupole analyzer (TSQ Quantum XLS, Thermo Fisher

190

Scientific, Bremen, Germany) which was operated in positive electron ionization mode

191

(EI+, 40 eV of electron energy) and in the SRM (selective reaction monitoring)

192

detection mode with a resolution of 0.7 Da peak width. The equipment was controlled

193

using the Xcalibur® data system. Injections were performed in programmable

194

temperature vaporization (PTV) mode (2 µL; 90 ºC, hold for 0.05 min, then to 200 ºC at

195

14.5 ºC/s, hold for 1 min, then to 300 ºC at 10 ºC/s, hold for 1.5 min, and then to 330 ºC

196

at 10 ºC/s, hold for 30 min; splitless time: 1.5 min) in a capillary VF-5ht column (15 m

197

x 0.25 mm i.d., 0.10 µm film thickness) purchased from Varian (Lake forest, CA). The

198

oven temperature was programmed from 90 ºC (2 min) to 160 ºC at a rate of 15 ºC/min,

199

then to 225 ºC at 4 ºC/min to 290 ºC at 7 ºC/min, and then to 310 ºC (10 min) at 10

200

ºC/min. Helium was used as the carrier gas at a constant flow rate of 1.2 mL/min. The

201

temperature of the transfer line and the MS source were set at 300 ºC and 240 ºC,

202

respectively. Collision gas (Ar) pressure was set to 1.0 mTorr for all the experiments.

203

Identification and quantification was always carried out by the isotopic dilution method,

204

based on the detection, at the appropriate chromatographic retention time, of the two

205

most abundant transitions of each native and

206

maintenance of the experimental ratio between these transitions within an appropriate

207

range.

13

C12-labeled BDE congener and the



Page 10 of 35

208

Quality control criteria.

209

All analysis followed the quality criteria such as blanks, recoveries, and parallel analysis

210

complied with analytical standards as recommended by the EU Commission in the

211

directive for measuring dioxins in food 47. A blank analysis in each set of analysis (three

212

analyses and one blank) was carried out. The amounts of BFR compounds detected in

213

the blanks were subtracted from the values obtained for the samples. BDE-47 was

214

present in all blank samples. To minimized interferences in blanks, all the glassware,

215

chemicals, solvents, and equipment used during extraction and clean-up procedures as

216

well as the instrumentation used were routinely checked. Besides, for maintaining the

217

quality of the methodologies applied, the working group participates, over time, in

218

different international quality control studies for the analysis of POPs in biological and

219

food matrices, including dairy products, eggs, chicken, fish, and meat samples 48.

220

Recovery rates of the labeled compounds added to samples before extraction step

221

ranged between 70 and 98% for

222

between 63 and 77% for 13C12-BDE 100 and from 74 to 87% for 13C12-BDE 153, which

223

complied with analytical standards recommended by the EU for measuring dioxins in

224

food (the recoveries of the individual internal standards shall be in the range of 60 to

225

120%) 47. In the case of soy lecithin sample, the recoveries obtained were slightly lower

226

(50% for 13C12-BDE 47, 48% for 13C12-BDE 99, 61% for 13C12-BDE 100, and 66% for

227

13

228

recoveries in the soy lecithin sample is the nature of the sample itself. This particular

229

sample was commercialized as a kind of pellet with a texture and physical properties

230

quite different from a lyophilized sample. However, considering that the isotopic

231

dilution technique was used, the results are corrected by labeled standards and the final

232

concentrations reported are accurate.

13

C12-BDE 47, from 62 to 73% for

13

C12-BDE 99,

C12-BDE 153). To the best of our knowledge, the probably reason for these lower


Page 11 of 35


233

RESULTS AND DISCUSSION

234

Method development and characterization.

235

The operational parameters of the MS were optimized in order to maximize the

236

response of the target compounds. Two ionization energies (40 and 70 eV), in

237

combination with various filament emission current (10, 25, and 50 µA) were tested.

238

The best results were obtained with a combination of 40 eV and 50 µA, which led to

239

less fragmentation of the molecular ions, especially those with a high bromination

240

degree, so they were set for the following experiments.

241

For the optimization of the multiple reaction monitoring (SRM) method, different

242

transitions were studied in order to select the most intense ones and, if possible, to

243

achieve the highest number of identification points 49. For PBDEs, the precursor ions

244

were [M]+ for tri- to hexa-BDEs, and [M-Br2]+ for hepta- to deca-BDEs. It should be

245

noted that, for hepta- to deca-BDEs, the intensity of molecular ion cluster was always

246

lower than the ion intensity resulting from the loss of two bromine atoms. This is due to

247

the high fragmentation suffered by the highly brominated congeners and this fact

248

increases with increasing bromine substituents. Thus, the loss of two bromine atoms

249

([M-Br2]+) for tri- to hexa-BDEs, and the loss of Br2 and COBr groups for hepta- to

250

deca-BDEs, were the most abundant transitions. Additionally, for hepta-BDEs, the loss

251

of COBr3 group from [M-Br2]+ was also monitored. Some other transitions were

252

registered, i.e. for penta-BDEs, the loss of COBr group from [M-Br2]+; for hexa-BDEs,

253

the loss of Br2 from [M-Br2]+; and for hepta-BDEs, the loss of Br2 from [M]+ and [M-

254

Br2]+, although the relative abundance, in comparison with those previously mentioned,

255

was lower in all cases. In the case of the two emerging BFRs, for BTBPE, the loss of

256

[C2H4Br]+ from the [M-C6H2Br3O]+ fragment cluster, which leads to [M-C8H6Br4O]+ as



257

product ion, was monitored. For DBDPE, the transition selected corresponded to [M-

258

C7H2Br5]+ as a precursor ion, losing one and two bromine atoms to obtain [M-C7H2Br6]+

259

and [M-C7H2Br7]+ as product ions, respectively. The MS/MS collision induced

260

dissociation (CID) energies and the ion transitions monitored were individually

261

optimized for each compound, ensuring their unambiguous identification. Table 1

262

summarized the final transitions selected and the optimum CID voltage for each

263

transition to take place. As shown, optimum values of the collision energy were found

264

to be normally around 20 eV for the low-brominated compounds, increasing for high-

265

brominated congeners until values such as 63 eV.

266

Finally, once the parameters affecting the MS/MS response were optimized, precision,

267

linearity, and instrumental limits of detection (iLODs) of the GC-QqQ(MS/MS) method

268

developed were studied for all the PBDEs and emerging BFRs investigated. The

269

obtained precision results were as follow: regarding areas, the repeatability (intraday

270

precision, RSD, %) was ≤ 12%, except for BDE-209, and the intermediate precision

271

(interday precision, RSD, %) was ≤ 20%, except for BTBPE, octa-BDEs and deca-

272

BDE. For ion transition ratios the repeatability was below 14% while the intermediate

273

precision was lower than 18%. Regarding linearity, the response of each native BFR

274

relative to that of its corresponding internal standard (13C12-labeled congeners) was

275

found to be linear in the tested range of 1 to 500 pg/µL (25 to 2500 pg/µL for BDE-209

276

and 10 to 5000 pg/µL for DBDPE), with correlation coefficients higher than 0.997.

277

With regards to iLODs, as can be seen in Table 2, values for tri- to octa-BDEs ranged

278

from 0.12 to 7.1 pg injected, for BDE-209 was 51 pg and for the two emerging BFRs,

279

BTBPE and DBDPE, were 1.1 and 20 pg injected, respectively. These values are in the

280

same range as those previously reported by other authors using different ionization

281

modes (EI

22,23,33,37,39-41,50,51

and ECNI

33,37

) and analyzers (ion trap detectors, ITD 22,50;


Page 12 of 35

Page 13 of 35


282

single quadrupole, Q 33; triple quadrupole, QqQ 37,39-41; and high resolution MS, HRMS

283

22,23,40,51

284

values for BDE-209 and emerging BFRs (see Table 2), which are compounds with

285

well-known analytical determination challenges. On the other hand, it should be

286

highlighted that the iLODs reported for GC-HRMS by Mackintosh et al., 2012

287

calculated using the SD of the lowest solution of the calibration curve, instead of S/N=3

288

as was done in the rest of the studies employing HRMS

289

procedure (the same followed in the present study) typically results in higher iLOD

290

values.

291

So, the analytical characteristics obtained in the present work indicate that GC-EI-

292

QqQ(MS/MS) is a suitable technique for BFRs determination in foods, providing an

293

acceptable precision, good linearity, iLODs in the low pg level, and enough sensitivity

294

and high selectivity for all target compounds. Besides, GC-EI-QqQ(MS/MS) provides

295

additional advantages in comparison to both EI-HRMS and ECNI-Q(SIM). For the

296

former, QqQ(MS/MS) possess lower costs of acquisition and maintenance along with

297

its ease of use, which makes this technique accessible to a larger number of laboratories.

298

And, for the latter, the low selectivity of the ECNI determinations is well known since

299

the most intense ions registered are bromine ions, instead of the specific transitions used

300

in QqQ(MS/MS).

301

The instrumental methodology developed was employed for the identification and

302

quantification of PBDEs three samples (egg yolk, breast milk, and cod liver oil)

303

belonging

304

Comparison on Dioxins in Food 2005 and 2006, Folkehelsa, Oslo, Norway). The results

305

were consistent with the consensus means given by the inter-laboratory organization

306

and the uncertainty of the measurements was lower than 15% in all cases.

). It is important to mention that some authors

to

different

international

37,51

interlaboratory

have reported higher iLOD

40

were

22,23,51

, and this calculation

exercises


(Interlaboratory


307

BFR concentrations in food samples enriched with omega-3 PUFAs.

308

Once characterized, the developed method was applied to the analysis of different

309

commercially available food samples enriched with omega-3 PUFAs. Concentrations

310

(median and range in pg/g fresh weight, f.w.) of each compound, as well as total BFRs

311

(expressed in both pg/g f.w. and pg/g lipid weight, l.w.) in the food samples studied are

312

summarized in Table 3. The values were calculated assuming that the concentration of

313

non-detected compounds is equal to zero. Lipid content of the samples is also included

314

in Table 3.

315

The highest ΣBFR concentrations were found in fish oil dietary supplements (median of

316

924, range of 111-6970 pg/g f.w.), followed by biscuits (median of 90, range of 15-165

317

pg/g f.w.), vegetable oil dietary supplement (46 pg/g f.w.), chicken eggs (median of 45,

318

range of 41-93 pg/g f.w.), cow's milk (median of 7.7, range of 3.8-8.6 pg/g f.w.) and soy

319

products (median of 1.6, range of 1.2-12 pg/g f.w.).

320

Figure 1 shows the BFR profiles of the different type of foodstuffs analyzed. The profile

321

of BFRs in cow’s milk and in biscuits was similar, being the most abundant compound

322

BDE-209 followed by BDE-47 and BDE-99 with median concentrations of 2.2, 1.6 and

323

0.63 pg/g f.w. and 72, 9.5 and 2.8 pg/g f.w., respectively. In both cases, the three

324

congeners account with more than 80% to the total BFR concentration. However, in

325

chicken eggs, the most abundant compound was DBDPE (54%) followed by BDE-47

326

(13%) and BDE-100 (9%) with median concentrations of 24, 12 and 8.4 pg/g f.w.,

327

respectively. In soy products, the profile of BFRs was led by BDE-47 (47%, median of

328

0.91 pg/g f.w.) followed by BDE-99 (25%, median of 0.40 pg/g f.w.). In regard to oil

329

dietary supplements, as can be seen in Figure 1, the BFR profile was different in fish

330

oils than in vegetable oil. The fish oil profile was dominated by BDE-47 (55%, median


Page 14 of 35

Page 15 of 35


331

of 472 pg/g f.w.) followed by BDE-99 (12%, median of 145 pg/g f.w.) and BDE-100

332

(11%, median of 101 pg/g f.w.), whereas the three most abundant BFRs in vegetable oil

333

were BDE-47 (37%), DBDPE (29%), and BDE-99 (21%) with concentrations of 17.2,

334

13.4 and 9.83 pg/g f.w., respectively. The high median contribution of BDE-209 in

335

cow’s milk (49%) and biscuits (44%), and BDE-47 in soy products (47%) and fish oil

336

dietary supplements (55%) is noteworthy. For the two emerging BFRs, DBDPE was

337

detected in all food groups except in soy products, and the most remarkable finding was

338

its high percentage of contribution in chicken eggs (54%) and vegetable oil (29%),

339

whereas in the rest of the samples its contribution was very low (lower than 1%). On the

340

other hand, BTBPE was present in all food groups but with percentages of contribution

341

lower than 4% in all cases. Figure S.1 (on Supplementary Information) shows, as an

342

example, the chromatograms corresponding to the transitions of hexa-BDEs, BTBPE,

343

BDE-209, and DBDPE in different food samples.

344

So, the results obtained in the present study are in agreement with the congeners

345

considered to be of primary interest by the CONTAM Panel 1. On the other hand, the

346

occurrence of DBDPE and BTBPE in some of the food groups analyzed suggests that

347

they have been increasingly used and consequently their levels should be monitored not

348

only in food, but also in humans, wildlife and any environmental compartment.

349

Comparison with other studies.

350

The results obtained in the present study have been compared with data previously

351

published for the same type of foodstuffs (chicken eggs, cow’s milk, biscuits, and oil

352

dietary supplements and other edible oils (olive and sunflower oils) and fats

353

(margarine)) sampled in Spain since 2000. Caution is necessary when comparing results

354

with values reported in the literature due to several parameters that can condition their



Page 16 of 35

355

calculation 17. In the different published studies there may be significant differences in

356

the sampling strategy, the number of BDE congeners studied, the calculation of results

357

in dry, lipid, or wet/fresh weight basis, the way in which concentrations are expressed

358

(upper bound, ND = LOD; medium bound, ND = LOD/2; lower bound, ND = 0), the

359

lipid weight calculation of the samples, and the year of sampling, which are among the

360

most important factors which influence the final results.

361

In most of the cases, the results obtained in the present study are in the same range or

362

slightly lower than those previously reported in Spanish surveys considering the same

363

types of foodstuffs. In the case of chicken eggs, previous total PBDE median

364

concentrations published varied from 58.3 pg/g f.w. for conventional eggs sampled in

365

2000

366

2005 17 and 2006 19, respectively, all of them being higher than the 45 pg/g f.w. median

367

concentration obtained in the present survey for chicken eggs enriched with omega-3

368

PUFAs. The same happen with commercial cow’s milk, the total PBDE median

369

concentrations were similar either in the enriched samples analyzed in the present study

370

(7.7 pg/g f.w.) or in conventional cow’s milk samples bought in Spain in 2000

371

pg/g f.w.), 2003-2005

372

samples, only one previous study

373

foodstuff, its mean concentration being (98.5 pg/g f.w.) slightly higher than the median

374

concentration found in enriched biscuits in the present study (90 pg/g f.w.). Regarding

375

oil dietary supplements, as it has been mentioned above, there is only one previous

376

study

377

supplements, the PBDE concentrations previously published were higher (620 pg/g

378

f.w.) than the levels found for the linseed oil dietary supplement analyzed in this study

379

(46 pg/g f.w.). On the other hand, for fish and mixed (fish plus vegetable) oil

16

to 73.5 and 94.8 pg/g f.w. (mean concentration) for samples collected in 2003-

20

17

(11.4 pg/g f.w.), and 2006 19

19

16

(13.2

(11.3 pg/g f.w.). For biscuits

reported PBDE concentrations for this type of

dealing with these types of samples. In the case of vegetable oil health


Page 17 of 35


380

supplements the opposite trend was observed, the present values being (924 pg/g f.w.)

381

higher than those previously reported in the literature (620 and 205 pg/g f.w. for fish

382

and mixed oils, respectively). It is important to highlight that in all studies,

383

concentration levels of PBDEs in fish and mixed oils were higher than those of

384

vegetable oils, and this difference could be explained by the origin of the oil. As has

385

been suggested in the literature

386

and biomagnification while PBDEs in vegetables occur from the deposition processes,

387

so no bioaccumulation or biomagnification is produced in vegetables, thus, lower

388

concentrations can be expected for this type of dietary supplements. Some authors have

389

also reported on PBDE concentrations in edible vegetable oils (olive and sunflower oils)

390

and fats (margarine) in the range of 119 to 569.3 pg/g f.w.

391

also higher than the concentrations found in the present study for vegetable oil dietary

392

supplements.

393

Taking into account the cautions mentioned above, it should be highlighted that the

394

study of the samples collected in 2003-2005

395

which BDE-209 (one of the three most abundant PBDE congeners) has been

396

determined. For the rest of the studies, only congeners with a bromination degree as

397

high as octa- have been quantified either as homologues families (from tetra- to octa-)

398

16,19,26

399

So, it seems that the PBDE concentrations have decreased in Spanish foodstuffs since

400

2000 until 2012, as expected due to bans or restrictions implemented in the EU, except

401

for the fish oil dietary supplements that present the highest concentrations (924 pg/g

402

f.w.) in this study.

403

In addition, a similar study was carried out in Canada in 2005-2006 comparing BFR

404

concentrations in chicken egg yolks from different origins (including, conventional and

20

, or as individual congeners

, PBDEs in fish suffer a process of bioaccumulation

20

17

16,17,19,26

, values which are

is the only one (in addition to this) in

(seven individual congeners from tetra- to octa-).



23

405

omega-3 enriched)

. In this case, the PBDE concentrations found were much higher

406

than those reported in the present study (median of 511 pg/g l.w.), being even higher for

407

conventional chicken eggs (5830 pg/g l.w.) than for omega-3 enriched samples (3250

408

pg/g l.w.). In addition, it should be pointed out that, in this Canadian study, 23 PBDE

409

congeners (from tri- to deca- bromination degree) have been detected, including the

410

most abundant ones (BDEs 47, 99, and 100). However, to the best of our knowledge,

411

this fact alone does not explain the high concentrations found that could be more related

412

with the country of origin. Another analogous study has been carried out in milks and

413

cheeses sampled in Italy in 2011 27 investigating the differences in POP levels between

414

conventional and omega-3 enriched samples. As for the Canadian and Spanish studies,

415

in most cases, the concentrations found in enriched samples were similar or slightly

416

lower than those obtained for conventional samples. So, considering all the above cases

417

mentioned, it could be concluded that, contrary to what occurs for other POPs (such as

418

PCDD/Fs and PCBs), samples enriched with omega-3 PUFAs do not show higher

419

PBDE concentrations than conventional samples do.

420

BFR intake.

421

Since fish oil pills, sold as a healthy food dietary supplement, showed the highest ΣBFR

422

concentrations, an assessment of the daily intake of BFRs according to the type of oil

423

consumed as dietary supplement and the daily dose of oil pills recommended was made.

424

The estimation of the BFR intake was calculated by multiplying the concentration of

425

ΣBFRs in each oil dietary supplement by the maximum recommended daily dose intake

426

(Table 4). For fish oil supplements the daily intake of BFRs ranged from 444 to 16171

427

pg/day. These results are within the intervals of values for PBDE daily intakes

428

published by other authors from different countries. Thus, for example, values ranging

429

from 1400 to 7800 pg/day were reported in Belgium 25, from 276 to 185000 pg/day in 18 ACS Paragon Plus Environment

Page 18 of 35

Page 19 of 35


24

, and from 100 to 44800 pg/day in Spain

20

430

Canada

431

levels of daily intake of BFRs published by other authors in Spain (23300 pg/day

432

9600 pg/day

433

present study, which was 37 pg/day for the linseed oil supplement.

434

On the other hand, the levels of PBDE intakes from fish or fish and shellfish

435

consumption published by other authors (29900 pg/day

436

pg/day

437

estimated from the consumption of the fish oil dietary supplements analyzed in this

438

study. This fact indicates that, the intake of BFRs via consumption of oil-based food

439

supplements, either fish or vegetable origin, would result in a lower BFR exposure than

440

normal fish consumption.

441

ABBREVIATIONS USED

442

ABS: acrylonitrile butadiene styrene; BFRs: brominated flame retardants; BTBPE: 1,2-

443

bis(2,4,6-tribromophenoxy) ethane; CID: collision induced dissociation; DBDPE:

444

decabromodiphenyl ethane; DHA: docosa-hexaenoic-acid; ECNI: electron chemical

445

negative ionization; EFSA: European Food Safety Authority; EI: electron ionization;

446

EPA: eicosa-pentaenoic-acid; EU: European Union; f.w.: fresh weight; GC: gas

447

chromatography; HIP: high impact polystyrene; HRMS: high resolution mass

448

spectrometry; iLODs: instrumental limits of detection; ITD: ion trap detector; l.w.: lipid

449

weight; MS: mass spectrometry; MS/MS: MS/MS tandem mass spectrometry; MSPD:

450

matrix solid phase dispersion; PBDEs: polybrominated diphenyl ethers; POPs:

451

persistent organic pollutants; PTV: programmable temperature vaporization; PUFAs:

452

poly unsaturated fatty acids; QqQ: triple quadruple analyzer; RSD: relative standard

453

deviation; SD: standard deviation; SIM: selected ion monitoring; S/N: signal to noise

19

, and 140 pg/day

25

, and 225000 pg/day

24

20

. Regarding vegetable oils, the 16

,

) were greatly higher than those estimated in the

16

, 26500 pg/day

19

, 23000

) were much higher than all values of BFR intakes



454

ratio; SPE: solid phase extraction; SRM: selective reaction monitoring; v:v:

455

volume:volume; w:w: weight: weight.

456

ACKNOWLEDGEMENTS

457

A. García-Bermejo wishes to thank Spanish Ministry of Education and Science for his

458

Ph.D. grant.

459

460

SUPPORTING INFORMATION

461

Figure S.1. Chromatograms corresponding to the transitions of hexa-BDEs, BTBPE,

462

BDE-209, and DBDPE in different food samples.

463


Page 20 of 35

Page 21 of 35


464

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Rapid Commun. Mass Spectrom. 2006, 20, 69-74.

623

(51) Kolic, T.M.; Shen, L.; MacPherson, K.; Fayez, L.; Gobran, T.; Helm, P.A.;

624

Marvin, C.H.; Arsenault, G.; Reiner, E.J. The analysis of halogenated flame retardants

625

by GC-HRMS in environmental samples. J. Chromatogr. Sci. 2009, 47, 83-91.

626

627

628

Founding sources: financial support was obtained from MICINN (projects AGL2009-

629

09733 and AGL2012-37201) and Community of Madrid (Spain), and European funding

630

from FEDER program (projects S2009/AGR-1464, ANALISYC-II and S2013/ABI-

631

3028-AVANSECAL).

632


Page 28 of 35

Page 29 of 35


633

FIGURE CAPTIONS

634

Figure 1. Percentages of contribution of individual BFR to the total BFR concentrations

635

in cow’s milk, chicken eggs, soy products, biscuits, fish and vegetable oil dietary

636

supplements.

637

638



Page 30 of 35

Table 1. Chromatographic segments, retention times, SRM transitions, and CID voltages selected for native BDEs and emerging BFRs in the GC-QqQ(MS/MS).

Segment No.

Time (min)

Compound

1

0.0 – 13.0

BDEs 17, 28

2

13.0 – 17.0

BDEs 47, 66

3

17.0 – 20.5

BDEs 99, 100

4

20.5 – 24.5

BDEs 153, 154

BDEs 183, 184, 191 5

24.5 – 28.0 BTBPE

6

28.0 – 31.0

BDEs 196, 197

BDE 209 7

31.0 - 45.0 DBDPE

Precursor ion

Product ion

CID (V)

406

246 (Q)

19

408

248 (q)

18

486

326 (Q)

18

488

328 (q)

21

564

404 (Q)

22

566

406 (q)

20

644

484 (Q)

23

642

482 (q)

24

564

455 (Q)

36

562

295 (q)

59

359

252 (Q)

23

357

252 (q)

26

642

482 (Q)

45

642

535 (q)

39

798

638 (Q)

52

800

640 (q)

63

485

325 (Q)

37

485

404 (q)

31

Q: quantification transition, q: confirmation transition


Page 31 of 35


Table 2. Instrumental limits of detection (iLODs in pg injected) for PBDEs and emerging BFRs reported in different studies using different ionization modes and MS analyzers. Ionization mode

Technique

Compounds analyzed

tri- to hexaBDEs

hepta-BDEs

octa-BDEs

BDE-209

BTBPE

DBDPE

Reference

EI

GC-QqQ(MS/MS)

14 PBDEs, BTBPE, DBDPE

0.12 - 1.9

1.9 - 3.8

7.1

51

1.1

20

This study

EI

GC-ITD(MS/MS)

10 PBDEs

0.28 - 1.6

5.2

-

-

-

-

(50)

EI

GC-ITD(MS/MS)

7 PBDEs

0.50 - 1.0

1.0

-

-

-

-

(22)

EI

GC-Q(SIM)

15 PBDEs

0.01 - 0.07

0.21 - 0.38

0.08 - 0.12

10.9

-

-

(33)

ECNI

GC-Q(SIM)

15 PBDEs

0.01 - 0.08

0.01 - 0.03

0.01 - 0.05

0.51

-

-

(33)

EI

GC-QqQ(SIM)


3.00 - 6.00

13.0

-

173

24.0

1000

(37)

ECNI

GC-QqQ(SIM)


0.3 - 7.00

0.40

-

3.00

0.6

6.00

(37)

EI

GC-QqQ(MS/MS)


0.40 - 16.0

3.00

-

100

26.0

200

(37)

EI

GC-QqQ(MS/MS)

28 PBDEs

0.1 - 1.25

0.35 - 0.75

2.10 - 5.00

2.50

-

-

(39)

EI

GC-QqQ(MS/MS)

41 PBDEs

0.07 - 4.0

2.0 - 10

12 - 30

41

-

-

(40)

EI

GC-QqQ(MS/MS)

8 PBDEs, DBDPE

0.11 - 3.57

12.5

-

16.3

-

6.25

(41)

EI

GC-HRMS

7 PBDEs

0.05

0.10

-

-

-

-

(22)

EI

GC-HRMS


-

-

-

-

10

100

(51)

EI

GC-HRMS

23 PBDEs

-

-

(23)

EI

GC-HRMS

41 PBDEs

-

-

(40)

0.05 - 13.9 7.0 - 20

7.0 - 12

31

ACS Paragon Plus Environment

8.0 - 16

85


Page 32 of 35

Table 3. BFR concentrations (median and range), expressed in pg/g fresh weight (f.w.), in food samples enriched with omega-3 PUFAs.

BFRs

Cow's Milk (n = 3)

Soy Products (n = 3)

Median Range Median BDE-17 0.017 0.013-0.043 0.0055 BDE-28 0.041 0.014-0.047 0.040 BDE-47 1.6 0.79-2.1 0.91 BDE-66 0.13 0.039-0.13 0.022 BDE-100 0.19 0.11-0.42 0.075 BDE-99 0.63 0.43-3.0 0.40 BDE-154 0.017 0.014-0.25 0.0030 BDE-153 0.11 0.066-0.40 0.038 BDE-184 ND ND BDE-183 0.24 0.18-0.24 0.0067 BDE-191 ND ND BTBPE 0.35 0.15-0.45 0.039 BDE-197 ND ND BDE-196 ND ND BDE-209 2.2 1.9-3.8 0.045 DBDPE 0.0036 0.0031-0.048 ND ΣPBDEs 7.2 3.6-8.3 1.6 ΣeBFRs 0.35 0.15-0.50 0.039 ΣBFRs 7.7 3.8-8.6 1.6 % lipid 2 2-5 2 Σ BFRs (pg/g l.w.) b 210 b 182-427 b 49 b a 2% for soy drinks (n = 2) and 82% for soy lecithin (n = 1) b ΣBFRs expressed in pg/g lipid weight (l.w.)

Range 0.0024-0.062 0.029-0.21 0.55-5.4 0.020-0.062 0.059-0.33 0.33-1.5 ND-0.017 0.0061-0.47 0.0044-0.97 0.019-2.9 ND-0.13 1.1-8.9 0.019-2.9 1.2-12 2 - 82 a 14-71 b

Chicken Eggs (n = 3) Median ND 0.071 12 0.064 8.4 4.2 3.0 2.3 ND 0.51 ND 0.43 ND ND 6.6 24 42 26 45 9 511 b

Range 0.028-0.17 2.5-19 ND-0.075 0.23-8.5 1.7-5.0 0.26-5.5 0.98-3.2 ND-0.32 ND-1.1 ND-1.8 ND-0.31 ND-0.37 3.7-11 1.5-50 15-43 1.5-50 41-93 8-9 491-1047 b

Biscuits (n = 2) Median 0.083 0.32 9.5 0.27 0.72 2.8 0.015 0.19 ND 0.59 ND 0.60 0.57 2.7 72 0.041 90 0.64 90 20 462 b

32


Range 0.063-0.10 0.26-0.38 9.2-9.8 0.17-0.38 0.72-0.73 2.6-3.0 ND-0.029 0.12-0.26 0.44-0.73 0.51-0.68 ND-1.1 ND-5.3 ND-144 ND-0.083 15-164 0.51-0.76 15-165 79-845 b

Fish oil dietary supplement (n = 6) Median Range 16 ND-46 50 0.53-275 472 65-4429 38 2.3-566 101 9.1-846 145 20-639 44 3.7-475 15 ND-144 ND 4.2 ND-20 ND 17 ND-204 ND ND ND 8.2 2.7-47 881 108-6945 33 2.7-151 924 111-6970 100 924 b 111-6970 b

Vegetable oil diet. supplement (n = 1) Median Range ND 1.9 17 0.60 1.2 9.8 ND 2.1 ND ND ND ND ND ND ND 13 33 13 46 100 46 b -

Page 33 of 35


Table 4. Estimated daily intake of BFRs and Omega-3 PUFAs associated with the ingestion of the different oils dietary supplements analyzed in the present study. Oil dietary supplements

BFR intake (pg/day)

Omega-3 intake (mg/day)

Cod

9182

-

Fish-1

16171

1800

Fish-2

444

2800

Salmon-1

912

-

Salmon-2

2453

684

Shark

466

-

Linseed

37

878



60

60

Cow's milk

Chicken eggs

50 % Contribution

% Contribution

50 40 30 20

40 30 20

10

10

0

0

BFRs

BFRs 50

50

Soy products

40 % Contribution

% Contribution

40 30 20

30 20

10

10

0

0

BFRs

BFRs 60 50

Biscuits

40

Fish oils

Vegetable oil % Contribution

% Contribution

30 40 30 20

20

10 10 0

0

BFRs

BFRs


Page 34 of 35

Page 35 of 35


PBDEs & BFRs


Applicability of Gas Chromatography (GC) Coupled to Triple

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