Simultaneous Determination of Acrylamide and 5

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New Analytical Methods

Simultaneous determination of acrylamide and 5hydroxymethylfurfural in heat processed foods employing EMR-Lipid as a new dispersive solid-phase extraction sorbent followed by liquid chromatography-tandem mass spectrometry Yousheng Huang, Chang Li, Huiyu Hu, Yuting Wang, Mingyue Shen, Shao-Ping Nie, Jie Chen, Maomao Zeng, and Ming-Yong Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05703 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Journal of Agricultural and Food Chemistry

1

Simultaneous

determination

2

5-hydroxymethylfurfural

3

EMR-Lipid as a new dispersive solid-phase extraction sorbent

4

followed by liquid chromatography-tandem mass spectrometry

in

of

heat

acrylamide

processed

foods

and

employing

5 6

Yousheng Huang1, 2, Chang Li1, Huiyu Hu1, Yuting Wang1, Mingyue Shen1*,

7

Shaoping Nie1, Jie Chen3, Maomao Zeng3, Mingyong Xie1*

8 9

1State

Key Laboratory of Food Science and Technology, China-Canada Joint Lab of

10

Food Science and Technology (Nanchang), Nanchang University, 235 Nanjing East

11

Road, Nanchang 330047, China

12

2Jiangxi

13

3State

14

14122, China

Institute of Analysis and Testing, Nanchang 330029, China

Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 2

15 16

* Corresponding author:

17

Mingyue Shen, PhD

18

Tel.: 0791-88304447-8328

19

E-mail address: [email protected]

20

Professor Mingyong Xie, PhD

21

Tel: +86 791 88305860

22

E-mail address: [email protected]

1

ACS Paragon Plus Environment


23

ABSTRACT: The goal of this study was to develop a method for simultaneous

24

determination of acrylamide (AA) and 5-hydroxymethylfurfural (5-HMF) in heat

25

processed foods by LC-MS/MS analysis. Several clean-up methods for QuEChERS

26

protocol were investigated and compared: (a) Dispersive solid phase extraction

27

(d-SPE) with Enhanced Matrix Removal-Lipid (EMR-Lipid); (b) d-SPE with PSA; (c)

28

without clean-up step; (d) clean-up with n-hexane. It's the first time that EMR-Lipid

29

sorbent has been used as d-SPE material to detect AA and 5-HMF in heat processed

30

foods, and among the four clean-up methods, EMR-Lipid method provided the best

31

clean-up of co-extracted matrix interferences and the highest extraction efficiency.

32

Validation experiments were carried out for the method of using EMR-Lipid as d-SPE

33

sorbent. Excellent linearity (R2 > 0.999) was achieved and the limits of detection

34

(LODs) of AA and 5-HMF were 2.5 μg/kg and 12.5 μg/kg, respectively. The

35

recoveries of AA and 5-HMF levels obtained were in the range of 87.3-103.3% and

36

83.2-104.3%, with precision (RSDs) of 1.2-6.8% and 1.4-7.4% (n=3), respectively.

37

The method is accurate and reliable and it was successfully applied to analyze the AA

38

and 5-HMF in 8 categories of Chinese heat processed foods.

39 40

KEYWORDS: EMR-Lipid; Acrylamide; 5-hydroxymethylfurfural; QuEChERS;

41

LC-MS/MS; Heat processed foods

2


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INTRODUCTION

43

A series of chemical reactions occur during heat processing of food, including

44

Maillard reaction, caramelization and lipid oxidation, which may result in both

45

desired and undesired effects on food quality

46

5-hydroxymethylfurfural (5-HMF) are two important contaminants produced during

47

heat processing, and they are receiving increasing attention due to their potential

48

toxicity and wide occurrence

49

genotoxic compound and has also been classified as a probable human carcinogen

50

(Group 2A) by International Agency Research on Cancer (IARC) 4. It is formed in

51

food during heating processes such as baking, frying, grilling, and roasting, with the

52

major reaction between amino group of asparagine and carbonyl group of reducing

53

sugars during heating at temperature higher than 120 °C

54

serious risk to human health, but there are concerns in the potential genotoxic and

55

carcinogenic

56

5-chloromethylfurfural 2, 9. It is formed by the caramelization of sugars under thermal

57

treatment and acidic catalysis, and reducing sugars can also generate 5-HMF through

58

the formation of 3-deoxyglucosone

59

both AA and 5-HMF, it is necessary to develop a simultaneous determination method

60

to improve the efficiency of risk assessment and control the quality of heat processed

61

foods.

62 63

properties

2, 3.

of

1,

2.

Acrylamide (AA) and

AA has been demonstrated to be a neurotoxic and

its

metabolites

10, 11.

5-8.

5-HMF does not pose a

5-sulfoxymethylfurfural

and

Since most of heat processed foods contain

In recent years, conventional methods for quantitation of AA and 5-HMF include gas chromatography (GC)

12, 13,

high-performance liquid chromatography (HPLC) 3



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64

14-16,

65

chromatography-tandem mass spectrometry (LC-MS/MS)

66

methods, LC-MS/MS was used as a powerful technique for the determination of AA

67

and 5-HMF alone or simultaneously in various food samples with high detection

68

sensitivity and without the derivatization step. However, to the authors’ best

69

knowledge, only one paper with regard to the simultaneous determination of AA and

70

5-HMF in food matrix (seafood) has been published

71

clean-up step was excluded for reducing recovery of 5-HMF by approximately 70%,

72

which could result in more matrix interference, decreased column lifetime and

73

increased instrument maintenance. Considering the necessities of purification of

74

extracts and lots of other heat processed foods containing AA and 5-HMF

75

simultaneously,

76

determination methods and broaden their application in more categories of food

77

matrices.

gas

chromatography-mass

further

studies

spectrometry

are

necessary

(GC-MS)

25.

to

17-19

20-24.

and

liquid

Among these

Moreover, in that study, the

investigate

simultaneous

78

The QuEChERS (acronym of quick, easy, cheap, effective, rugged and safe)

79

method, including extraction and dispersive solid phase extraction (d-SPE) clean-up

80

step, has been developed and used for the extraction of AA or 5-HMF in the last few

81

years

82

difference of AA and 5-HMF in polarity and solubility, the ability of d-SPE sorbent to

83

clean up interfering compounds from food matrices without sacrifice of target

84

compounds’ recoveries is very important for the simultaneous determination of AA

85

and 5-HMF. Primary secondary amine (PSA) is a well-known sorbent employed in

17, 20, 25, 26.

For the complexity of food matrices such as high lipids and the

4


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86

the QuEChERS method when removal of lipids from food matrices is necessary 27, 28,

87

but the fact that addition of PSA could reduce the recovery of AA and 5-HMF was

88

also reported by researchers 20, 25. Moreover, several new d-SPE sorbents such as sol–

89

gel hybrid methyltrimethoxysilane–tetraethoxysilane and magnetic graphene sol–gel

90

hybrid were used in the analysis of AA in heat processed food in recent years

91

However, the effectiveness of d-SPE sorbents to clean up co-extracts for the

92

simultaneous determination of AA and 5-HMF in food matrices has never been

93

investigated before.

17, 29.

94

Due to the high-fat content of most heat processed foods, it is important to remove

95

lipids from the extract before LC-MS/MS analysis. Recently, a vendor introduced a

96

unique product known as “Enhanced Matrix Removal-Lipid” (EMR-Lipid). The

97

manufacturer claims that lipids are removed selectively by EMR-Lipid from

98

QuEChERS extracts of fatty foods such as avocado and animal tissues, without loss of

99

target substance ， such as pesticides, veterinary drugs, or PAHs

30-32.

Preliminary

100

results with the novel sorbent material EMR-Lipid are promising for removing

101

unwanted matrix interferences from the extract without loss of the target compounds

102

33-36.

103

property interests, but the extraction mechanism is said to involve both size exclusion

104

and hydrophobic interactions. Lipids with long-chain hydrocarbons are trapped via

105

association with the EMR-Lipid structure. AA and 5-HMF are hydrophilic molecules,

106

which may be separated from interfering compounds such as lipids in heat processed

107

foods by EMR-Lipid, similar to pesticides determined in fatty foods. It could be a

The composition of EMR-Lipid has not been disclosed yet due to intellectual

5



108

good try to select EMR-Lipid as d-SPE sorbent of QuEChERS method. However, to

109

the authors' best knowledge, no previous study has been reported for using

110

EMR-Lipid sorbent as d-SPE material to detect AA and 5-HMF.

111

Taking all these points into account and considering the importance of

112

simultaneous determination of AA and 5-HMF, the objective of the present study is to

113

develop a convenient, sensitive and accurate quantitative method for the simultaneous

114

determination of AA and 5-HMF in heat processed foods. The performance of d-SPE

115

with EMR-Lipid was compared with those of three other clean-up methods (d-SPE

116

with PSA, without clean-up step and clean-up with n-hexane) in QuEChERS protocol.

117

Moreover, the finally selected method was validated and used to assess the levels of

118

AA and 5-HMF in eight different categories of heat processed foods in China.

119

MATERIALS AND METHODS

120

Samples

121

Eight categories of commercially available foods including fried potato chips,

122

biscuits, bread, deep-fried stick (you-tiao), fried peanuts, fried fragrant taro, fried

123

chips made with rice and peanuts (yueliangba) and coffee beverage were purchased

124

from local supermarkets in Nanchang, Jiangxi Province, China. Number of samples

125

(Sample ID) for each category is shown in Supplementary Table 1. All the samples

126

were ground into powder by a homogenizer and stored at -20ºC before analysis.

6


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127


Chemicals and Materials

128

AA, 5-HMF, 13C3-AA and 13C6-HMF were purchased from Sigma-Aldrich (USA).

129

HPLC-grade acetonitrile, methanol, n-hexane and formic acid were obtained from

130

Merck Company (Darmstadt, Germany). Distilled water used for extraction and as

131

HPLC mobile phase was purchased from Watsons Water Co., Ltd (Guangzhou,

132

China). Sodium chloride (NaCl) and anhydrous magnesium sulphate (MgSO4) were

133

obtained from Sinopharm chemical reagent co.ltd (Shanghai, China). Bond Elut

134

EMR-Lipid was purchased from Agilent Technologies (Santa Clara, USA) consisting

135

of 1 g EMR-Lipid material in a 15 mL polypropylene tube and 2 g EMR-Polish salts

136

(anhydrous MgSO4:NaCl, 4:1, w/w) in a second 15 mL centrifuge tube. PSA and

137

anhydrous MgSO4 for QuEChERS were purchased from Agilent Technologies

138

(Shanghai, China).

139

Standards Preparation

140

Stock solutions of AA, 5-HMF,

13C

3-AA

and

13C

6-HMF

were prepared by

141

weighing 10 mg of each analyte into a 10 mL volumetric flask and adding water to

142

obtain the final volume of 10 mL. The mixed working standard solutions were

143

obtained by dilution of stock solutions with water.

144

LC-MS/MS Analysis

145

An Agilent 1200 Series high performance liquid chromatograph (HPLC) system

146

(Agilent Technologies Inc, USA) equipped with a binary pump, a solvent degasser, an

147

autosampler and a thermostatically controlled column apartment was used for 7



148

chromatographic analysis. Separation of the analytes was achieved on a Synergi

149

Hydro-RP column (150 × 2 mm i.d., 4 µm, Phenomenex, USA) with a column

150

temperature of 30 °C. The binary solvent system consisted of acetonitrile (A) and

151

0.1% (V/V) aqueous formic acid solution (B). The gradient profile of the binary pump

152

was started at 95% B (0 min), decreased to 20% B from 0 to 1.0 min, then maintained

153

at 20% B from 1.0 min to 3 min, finally, back to 95% B at 3.1 min, and equilibrating

154

for 5 min. The flow rate was 0.3 mL/min, and the injection volume was 5.0 μL.

155

The MS/MS detection was performed on an Agilent 6410 Triple Quadrupole mass

156

spectrometer (QQQ) (Agilent Technologies, USA), and the electrospray ionization

157

(ESI) source was operated in positive mode and data was acquired using multiple

158

reaction monitoring (MRM) mode. Each standard solution (1.0 μg/mL) was directly

159

injected into the QQQ to obtain the optimal parameters of precursor ion, product ion,

160

fragmentor voltage and collision energy (CE) (Table 1). The dwell time for all MRM

161

transitions of the analytes was set to 40 ms. The other working parameters of the mass

162

spectrometer were as follows: nebulizer gas: nitrogen; drying gas temperature: 350℃;

163

drying gas flow rate: 10 L/min; nebulizer pressure: 40 psi; capillary voltage: 4000 V.

164

Both MS1 and MS2 quadrupoles were maintained at unit resolution. MassHunter

165

Workstation (version B.01.03) (Agilent, Lake Forest, CA, USA) was used to control

166

the LC-MS/MS system and process the data.

167

Sample Preparation

168

1.0 g of ground sample was weighed into a 50 mL centrifuge tube, and spiked with

169

internal standards of 13C3-AA (250 μg/kg) and 13C6-HMF (1000 μg/kg), then 3 mL of 8


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170

distilled water was added. After standing for 20 min, 10 mL of acetonitrile was added

171

to sample and the mixture was then shaken by an IKA Vortex Genius 3 shaker (IKA,

172

Staufer, Germany) for 2 min. Then four versions of clean-up methods were compared:

173

(a) d-SPE with EMR-Lipid. The tubes were centrifuged at 4400 g for 5 min by an

174

Anke TDL-5-A low-speed centrifuge (Anke, Shanghai, China). Then 5.0 mL of the

175

supernatant was collected and added into a 15 mL d-SPE tube containing EMR-Lipid

176

sorbent and 3 mL water. Subsequently, the mixture was shaken vigorously for 1 min

177

and then centrifuged at 4400 g for 5 min. After that, the above extract (5 mL) was

178

transferred to a 15 mL EMR-Polish tube containing 2 g salts (anhydrous MgSO4:

179

NaCl, 4:1, w/w). The mixture was shaken vigorously for 1 min and then centrifuged

180

at 4400 g for 5 min. 2.0 mL of supernatant was transferred into a 10 mL glass test

181

tube.

182

(b) d-SPE with PSA. The prepared mix of QuEChERS pouch, composed of 4.0 g of

183

MgSO4 and 1.0 g of NaCl, was added to the 50 mL tube containing acetonitrile/water

184

extract and the mixture was shaken vigorously for 2 min. The tube was then

185

centrifuged at 4400 g for 5 min. For the clean-up step, 5.0 mL supernatant was

186

transferred to a centrifuge tube containing 250 mg of PSA and 750 mg of anhydrous

187

MgSO4. The tube was then shaken vigorously for 1 min and centrifuged at 4400 g for

188

5 min. 2.0 mL of supernatant was transferred into 10 mL a glass test tube.

189

(c) Without clean-up step. The extraction method was the same as version b and the

190

clean-up step was skipped. 2.0 mL of supernatant was transferred into a 10 mL glass

191

test tube. 9



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192

(d) Clean-up with n-hexane. The extraction method was the same as version b with

193

a different clean-up step described as follows. Instead of clean-up with PSA, 5 mL

194

n-hexane was added to the tube of 5.0 mL supernatant, then the mixture was vortexed

195

for 1 min and centrifuged at 4400 g for 5 min, afterward, the n-hexane layer was

196

discarded. The lower layer of acetonitrile (2 mL) was transferred into a 10 mL glass

197

test tube.

198

For each version, the solution of extract in the 10 mL glass test tube was dried by a

199

gentle nitrogen stream in a water bath at 40 ℃, then residue was re-dissolved in 1.0

200

mL water and filtered through a 0.22 μm PES membrane (Anpel, Shanghai, China),

201

for analysis by LC–MS/MS. The extraction and clean-up method for each sample was

202

carried out in triplicate. The detailed workflow for the best method (version a) is

203

shown in Fig.1.

204

Analyte loss during extraction (ALDE)

205

The extraction efficiency was evaluated by ALDE. As analytes and their internal

206

standards had the same behavior during sample preparation, 13C3-AA (250 μg/kg) and

207

13C

208

Then peak areas of internal standards in extracted sample solution were compared to

209

those of internal standard solution at the same concentrations (n=3). The ALDE was

210

calculated as the following formula:

211

ALDE (%) = 1 -

6-HMF

(1000 μg/kg) were added to fried potato chips before sample preparation.

(

peak areas of internal standards in extracted sample solution peak area of internal standard solution

10


)

× 100.

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212


Validation of the method

213

Validation experiments were carried out for the method of using EMR-Lipid as

214

d-SPE sorbents. Calibration curves were built by standard solutions at six different

215

concentration levels (5, 10, 20, 50, 100, 200 ng/mL for AA and 20, 40, 80, 200, 400,

216

800 ng/mL for 5-HMF), containing the internal standards at 50 ng/mL and 200

217

ng/mL, respectively. Each standard solution was analyzed three times and the average

218

peak area ratio between analyte and internal standard was plotted against the

219

corresponding concentration ratio of analyte to internal standard. Recovery of

220

analytes in eight different food categories was obtained by analyzing samples spiked

221

with standard containing AA (25, 100, 200 and 500 μg/kg) and 5-HMF (400, 800 and

222

2000 μg/kg) and their internal standards (250 μg/kg for AA and 1000 μg/kg for

223

5-HMF), and each recovery level was determined in triplicate. The recovery for AA

224

and 5-HMF was calculated as follows:

225

Recovery (%) =the amount of analytes added × 100.

226

The amount of analytes tested was calibrated by the calibration curves mentioned

227

above.

228

Statistical Analysis

the amount of analytes tested

229

Analysis of variance (ANOVA) was performed using SPSS software (version

230

19.0). The results were subjected to Tukey's test and P-value < 0.05 was considered

231

statistically significant.

11



232

RESULTS AND DISCUSSION

233

LC-MS/MS Optimization

234

The instrument parameters were optimized based on previous studies on the 20, 25.

235

analysis of AA and 5-HMF

Positive electrospray mode was proved to be the

236

most sensitive mode for the analytes and internal standards. All parameters were

237

optimized to obtain the strongest responses for the ion [M+H]+ of analytes. For LC

238

separation, a Synergi Hydro-RP column (150 × 2 mm; 4 µm) was selected. Its

239

property of polar endcapping enabled to provide acceptable retention and peak shape

240

for both AA and 5-HMF. In the final method, 95% aqueous solution containing 0.1%

241

formic acid and 5% acetonitrile was used as initial mobile phase for elution. The

242

chromatograms of analytes and internal standards are shown in Fig.2. Considering the

243

co-extract in the sample, a stronger mobile phase with high percentage of acetonitrile

244

was necessary to avoid the accumulation and eventually peak co-elution in the

245

following analysis.

246

Evaluation of different clean-up methods

247

The objective was to develop a QuEChERS pre-treatment method with suitable

248

clean-up method to extract AA and 5-HMF from various heat processed foods that

249

would be compatible with subsequent LC-MS/MS analysis. As starting points in the

250

method development, an addition of water was necessary to facilitate AA and 5-HMF

251

extraction from food samples, followed by the addition of acetonitrile as an

252

appropriate extraction solvent. In this study, several clean-up methods were 12


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253

investigated, including two d-SPE methods using EMR-Lipid product and PSA as

254

d-SPE sorbent materials respectively and a method of liquid-liquid extraction by

255

adding n-hexane. Samples without clean-up step were also compared. EMR-Lipid is a

256

novel clean-up sorbent for dispersive solid phase extraction employed for highly

257

selective matrix removal, especially for high-fat samples, incorporated in an initial

258

QuEChERS-based extraction step

259

efficiency of different clean-up methods were evaluated by using fried potato chips as

260

representative matrix. The results were discussed in the following text.

30, 31, 37.

The extract cleanliness and extraction

261

Extract cleanliness. Firstly, as an attempt to estimate the extract cleanliness of

262

different clean-up methods, full-scan total ion chromatograms (TICs) of potato chip

263

extracts obtained from different clean-up methods were overlapped for comparison

264

purposes. As shown in Fig.3, TICs obtained from the same potato chip matrix showed

265

that d-SPE with EMR-Lipid was the most effective clean-up method with minimum

266

amounts of matrix interferences existing in the final extract compared to other three

267

versions of clean-up method. Several studies also found that EMR-Lipid method was

268

more effective to clean up co-extracts during the determination of pesticide residues

269

and environmental contaminants in food matrix 34, 36. Compared with TICs of extracts

270

obtained by methods of clean-up with n-hexane and without clean-up step, the TIC of

271

the extract pretreated with PSA was close to that of the extract pretreated with

272

EMR-Lipid, which showed that the cleaning ability of PSA was comparable to that of

273

EMR-Lipid. PSA is a commonly used sorbent in QuEChERS method to remove fatty

274

acids, sugars and some pigments. Dunovska et al.

38

reported that PSA significantly

13



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275

reduced the matrix co-extracts (mainly free fatty acids) during the determination of

276

AA in food. However, we found that the utilization of PSA resulted in the loss of

277

analytes in the extraction step, especially for 5-HMF, as described below.

278

Moreover, as shown in Fig.3, it was obvious that co-extractive impurities of peak 1

279

and peak 2 are co-eluted with AA (1.9 min) and 5-HMF (3.9 min), which may cause

280

matrix effect and affect accuracy of determination. However, their peak areas in

281

EMR-Lipid method were minimum compared to those in other methods. For the

282

LC-MS/MS analysis of AA (Fig.4A), an interference peak having the retention time

283

of about 1.4 min was found, which could be the interference of amino acid valine

284

generating m/z 72 product ion in samples typically analyzed for AA

285

interference peak area of extracts obtained by clean-up method of n-hexane and

286

without clean-up step were higher than that of extracts obtained by d-SPE methods

287

(PSA and EMR-Lipid), showing the impurity was not removed sufficiently by method

288

of n-hexane or without the clean-up step.

39.

The

289

Extraction efficiency. In order to evaluate extraction efficiency, the peak area of

290

analytes in the same fried potato chip pretreated with four clean-up methods was

291

compared in Figure 4, and ALDE was also calculated. For AA, as shown in Fig.4A-B,

292

the peak area of AA in sample pretreated with EMR-Lipid is higher than those in

293

samples pretreated with other clean-up methods, and the ALDE of EMR-Lipid

294

method was less than those of other clean-up methods, demonstrating the use of

295

d-SPE with EMR-Lipid has the best extraction efficiency. The extract cleanliness of

296

d-SPE with PSA was close to that of the EMR-Lipid method as described above, 14


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297

however, PSA resulted in a 20% rise of analyte loss during extraction of AA

298

compared to EMR-Lipid, which was in agreement with previous study 20. Moreover,

299

although d-SPE sorbents have not been used in the method of n-hexane and without

300

clean-up step, the ALDE of these two methods were still higher than that of the

301

EMR-Lipid method.

302

For 5-HMF, the results were similar to those of AA. As shown in Fig.4C-D, the

303

peak area of 5-HMF in sample pretreated with EMR-Lipid is also higher than those in

304

samples pretreated with other clean-up methods, and the ALDE of EMR-Lipid

305

method was less than those of other clean-up methods. It was notable that the highest

306

ALDE (94%) was obtained when d-SPE with PSA was used, so most of 5-HMF was

307

lost when clean-up with PSA. Qin et al.

308

the 5-HMF loss by approximately 70% during extraction. For this reason, the clean-up

309

step of QuEChERS extraction was excluded in his study. Therefore, PSA is not a

310

suitable sorbent to clean up the matrix interferences for 5-HMF analysis.

25

also found that the addition of PSA made

311

Taking these results into account, it was found that clean-up method using

312

EMR-Lipid as d-SPE sorbent enabled to guarantee good extraction efficiency while it

313

could also ensure efficient and robust clean-up to remove unwanted matrix

314

interferences, especially lipids, from the extract, while increasing the overall

315

performance of the method. Therefore, EMR-Lipid was chosen as clean-up sorbent

316

for AA and 5-HMF determination in the heat processed foods. Validation experiments

317

were carried out for the selected method.

15



318

Page 16 of 39

Method Validation

319

Linearity and sensitivity. Considering the different levels of AA and 5-HMF in

320

most of the samples, different linear range of these two analytes was obtained.

321

Excellent linearity was achieved with a correlation coefficient (R2) ＞0.999 for both

322

AA and 5-HMF. The limits of detection (LOD) and quantification (LOQ) based on a

323

signal-to-noise ratio (S/N) of 3:1 and 10:1 were determined. In our study, the LODs of

324

AA and 5-HMF were 2.5 and 12.5 μg/kg, while the LOQs of AA and 5-HMF were

325

8.3 and 41.5 μg/kg, respectively.

326

Recovery and precision. As shown in Table 2, observed recoveries of AA and

327

5-HMF were in the range of 87.3-103.3% and 83.2-104.3% in 8 categories of heat

328

processed food samples, with RSDs ranging from 1.2% to 6.8% and 1.4% to 7.4%,

329

respectively. In comparison with the previous studies reported

330

precision of this analytical method were satisfactory.

20, 25,

the recovery and

331

Comparison with previous studies. As shown in Table 3, the LOD obtained in the

332

current study using EMR-Lipid as d-SPE sorbent is compared with those published in

333

previous studies using other preparation methods such as SPE and QuEChERs with or

334

without d-SPE. For AA, the sensitivity of this developed method is higher than the

335

results obtained using d-SPE with hybrid sol–gel MTMOS–TEOS and SPE with

336

mixture of C18, SCX and SAX sorbents 17, 21, and is comparable to that of QuEChERs

337

without d-SPE clean-up step

338

d-SPE sorbent gives the lowest LOD, as finally only 50 μL acetonitrile was added to

339

re-dissolve the residue for LC-MS/MS analysis, which highly concentrated analytes in

20.

The method using Fe3O4@G-TEOS-MTMOS as

16


Page 17 of 39


29.

340

sample solutions

For 5-HMF, the sensitivity of this developed method is

341

comparable to those of the SPE method with HLB and ENV+

342

sensitivity of this developed method is lower than the results of QuEChERs without

343

d-SPE clean-up step used for simultaneous determination of AA and 5-HMF in

344

seafood matrix 25. As the clean-up step was excluded in that study, it could result in

345

more matrix interference, decreased column lifetime and increased instrument

346

maintenance. In our study, the proposed QuEChERS with EMR-Lipid as d-SPE

347

sorbent offered a simple approach to efficiently clean up co-extracts for the

348

simultaneous determination of AA and 5-HMF in a variety of heat-processed foods.

349

Analysis of real samples

19, 24.

Moreover, the

350

The developed method was applied to determine levels of AA and 5-HMF in a

351

variety of commercially available heat processed foods in the Chinese market. Eight

352

categories of frequently-consumed foods including fried potato chips, biscuits, bread,

353

you-tiao, fried peanuts, fried fragrant taro, yueliangba and coffee beverage were

354

analyzed. The mean levels, minimum values and maximum values of AA and 5-HMF

355

in 8 categories of foods are shown in Table 4. AA was detected in the samples at

356

concentrations ranging from < LOD to 633.3 μg/kg and the levels fluctuated wildly

357

even if in the same category except for bread and coffee beverage. The mean AA

358

levels of 8 categories of food samples were in the order of potato chip > fragrant taro

359

chip > biscuit > yueliangba > you-tiao > bread > peanut > coffee beverage. Among all

360

food samples analyzed, the AA levels of 4 samples (two potato chip samples, one

361

biscuit sample and one fragrant taro chip sample) exceeded 300 μg/kg, while AA 17



Page 18 of 39

362

levels were below the LOD in 9 samples (one biscuit sample, three peanut sample and

363

all of coffee beverage samples). 18 samples (36% of total) contained AA at

364

concentrations of 100-300 μg/kg, while 19 samples (38% of total) contained AA at

365

concentrations lower than 100 μg/kg. These AA levels of potato chip, biscuit, peanut,

366

bread and you-tiao were in agreement with the previous studies reported

367

Taking potato chip as an example, we found the AA concentrations ranged from 44.8

368

μg/kg to 633.3 μg/kg with a mean value of 251.0 μg/kg, slightly lower than those

369

reported by Oroian et al. 43 in Romanian, ranging from 18 μg/kg to 1782 μg/kg, with a

370

mean value of 600 μg/kg. Bertuzzi et al.

371

ranging from 92 μg/kg to 1722 μg/kg, with a mean value of 803 μg/kg. It was notable

372

that the AA levels were below the LOD in 5 coffee beverage samples. Andrzejewski

373

et al.

374

those in ground coffee and instant coffee, ranging from 6 to 16 ng/mL (close to the

375

LOD). The AA loss in brewed coffee may be due to the dilution and brewing of

376

coffee. Moreover, to the best of our knowledge, no data about levels of AA in fragrant

377

taro chip and yueliangba was reported before, and our data could provide useful

378

information for the consumption and risk assessment of these foods.

44

42

20, 29, 40-43.

reported AA levels of potato chip in Italy

reported the levels in brewed coffee (coffee beverage) were much less than

379

The 5-HMF was detected in all the samples at concentrations ranging from 0.3 to

380

56.3 mg/kg and the levels also fluctuated wildly even if in the same category (Table

381

4). It was necessary that some samples containing high concentrations of 5-HMF

382

deserved an additional dilution of the extract before LC-MS/MS analysis in order to

383

obtain a response able to be quantified within the linear calibration range. The mean 18


Page 19 of 39


384

5-HMF levels of 8 categories of food samples were in the order of yueliangba >

385

coffee beverage > bread > biscuit > potato chip > fragrant taro chip > peanut >

386

you-tiao. Among all food samples analyzed, the 5-HMF levels of 8 samples (two

387

biscuit samples, one bread sample, two yueliangba samples and three coffee beverage

388

samples) exceeded 10 mg/kg. 32 samples (64% of total) contained 5-HMF at

389

concentrations of 1.0-10 mg/kg, while 10 food samples (20% of total) contained

390

5-HMF at concentrations lower than 1.0 mg/kg. These 5-HMF levels of potato chip,

391

biscuit, bread and coffee were in the same order of magnitude as those reported by

392

previous studies

393

concentrations ranged from 7.3 mg/kg to 23.5 mg/kg with a mean value of 5.2 mg/kg,

394

similar to those found by Ameur et al. 47, which ranged from 0.5 mg/kg to 74.6 mg/kg

395

with a mean value of 24.1 mg/kg, and slightly lower than those reported by

396

Delgado-Andrade et al. 48, which ranged from 3.1 mg/kg to 182.5 mg/kg with a mean

397

value of 14.4 mg/kg. Moreover, as mentioned above, to the best of our knowledge, no

398

data about levels of 5-HMF in you-tiao, peanut, taro chip and yueliangba was reported

399

before, and our data could provide useful information for the consumption and risk

400

assessment of these foods.

2, 19, 45-48.

For instance, for biscuits, we found the 5-HMF

401

402

ABBREVIATIONS USED

403

AA, Acrylamide; 5-HMF, 5-hydroxymethylfurfural; GC, gas chromatography; HPLC,

404

high-performance liquid chromatography; GC-MS, gas chromatography-mass 19



405

spectrometry; LC-MS/MS, liquid chromatography–tandem mass spectrometry;

406

QuEChERS, acronym of quick, easy, cheap, effective, rugged and safe; d-SPE,

407

dispersive solid phase extraction; PSA, Primary secondary amine; EMR-Lipid,

408

Enhanced Matrix Removal-Lipid; ALDE, Analyte loss during extraction.

409

ACKNOWLEDGEMENTS

410

The authors acknowledge the financial supports from the National Key Research

411

and Development Program of China (2017YFC1600405).

412

Notes

413

414 415 416 417 418

The authors declare no conflicts of interest.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. The sample ID and levels of AA and 5-HMF for each heat processed food sample (Supplementary Table 1)

419

20


Page 20 of 39

Page 21 of 39


420

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48. Delgado-Andrade,

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Page 29 of 39


580

Figure captions

581

Figure 1. Schematic diagram of the best extraction method (version a)

582

Figure 2. LC-MS/MS chromatograms of analytes and internal standards

583

Figure 3. Overlapped total ion chromatograms of potato chip extracts obtained with

584

different clean-up methods

585

Figure 4. Overlapped LC-MS/MS chromatogram and ALDE of different clean-up

586

methods (A. Overlapped LC-MS/MS chromatogram of AA in the same fried potato

587

chip; B. ALDE of AA calculated via the peak area of 13C3-AA; C. Overlapped

588

LC-MS/MS chromatogram of 5-HMF in the same fried potato chip; D. ALDE of

589

5-HMF calculated via the peak area of 13C6-HMF)

29



590

Figure 1

591 592

30


Page 30 of 39

Page 31 of 39

593


Figure 2

594 595

31



596

Figure 3

597 598

32


Page 32 of 39

Page 33 of 39

599


Figure 4

600

33



Page 34 of 39

602

Tables

603

Table 1. Mass spectrometric settings for MRM of the analytes and internal standards Compounds

Precursor ion

Product ion

Fragmentor(eV)

CE(eV)

75

58

40

10

72

55 (Q)

40

10

72

44 (C)

40

20

133

86

75

15

127

81 (C)

75

15

127

53 (Q)

75

20

13C

3-AA

AA 13C

6-HMF

5-HMF

604 605

Q: quantification ion; C: confirmation ion.

606

Table 2. Recoveries and RSDs of AA and 5-HMF in 8 categories of heat processed

607

food samples (n=3) Sample

Potato chips

Biscuits

Bread

You-tiao

Peanut

Taro chips

AA

5-HMF

Spiked(μg/kg)

Recovery(%)

RSD (%)

Spiked(μg/kg)

Recovery(%)

RSD (%)

25

90.9

4.7

--

--

--

100

95.7

2.0

400

98.9

5.8

200

95.8

4.0

800

88.6

6.2

500

98.3

4.7

2000

84.3

3.8

25

94.5

6.7

--

--

--

100

96.3

4.9

400

104.3

2.2

200

92.3

1.9

800

98.7

4.0

500

98.1

3.0

2000

91.3

6.8

25

88.7

5.2

--

--

--

100

94.3

4.6

400

90.4

5.4

200

89.7

3.3

800

83.4

7.4

500

99.8

3.1

2000

86.3

6.3

25

103.3

6.8

--

--

--

100

87.8

4.6

400

96.3

5.7

200

88.7

2.0

800

102.3

4.5

500

92.8

1.2

2000

91.5

3.6

25

87.3

2.9

--

--

--

100

92.7

4.0

400

98.9

5.8

200

94.7

1.8

800

100.9

5.1

500

96.4

2.5

2000

91.0

3.5

25

101.9

3.3

--

--

--

34


Page 35 of 39


Yueliangba

100

88.7

4.7

400

88.7

5.5

200

89.0

5.5

800

101.3

3.2

500

90.6

3.9

2000

85.7

5.5

25

94.6

3.3

--

--

--

100

96.3

5.3

400

86.9

3.9

200

93.5

1.5

800

96.7

1.4

500

88.6

4.1

2000

83.2

6.6

25

100.6

3.8

--

--

--

Coffee

100

97.7

2.4

400

87.8

2.9

beverage

200

89.5

2.5

800

83.8

6.9

500

88.0

2.7

2000

84.9

3.4

608

35



609

Page 36 of 39

Table 3. LOD of AA and 5-HMF in the current study compared to other studies Sample

Sample preparation method

LOD of AM (μg/kg)

LOD of 5-HMF (μg/kg)

Detector

Reference

Potato chip, biscuit, bread,fragrant taro chip, yueliangba, you-tiao, peanut and coffee beverage

d-SPE with EMR-Lipid

2.5

12.5

LC-MS/MS

Current study

Fried potato, fried eggplant, minnan and gorrasa

d-SPE with hybrid sol–gel MTMOS–TEOS

9.1-12.8

/

GC-MS

Omar et al.17

Boiled potato, fried potato with bright-fleshed, sweet potato, cheese snack, banana chips, fried eggplant, bread and potato chips

d-SPE with Fe3O4@G-TEOS-MTMOS

0.061-2.89

/

GC-MS

Rashidi Nodeh et al.29

SPE with mixture of C18, SCX and SAX sorbents

5

/

LC-MS/MS

Bortolomeazzi et al. 21

Dried fruits and edible seeds

QuEChERs without d-SPE clean-up step

2

/

LC-MS/MS

De Paola et al.20

Baby Foods

SPE with HLB

/

5

LC-MS/MS

Gökmen et al.24

Biscuits, breakfast cereals, orange juice, honey and jam

SPE with ENV+

/

12

GC-MS

Teixido et al.19

Thermally processed seafood

QuEChERs without d-SPE clean-up step

0.22

0.27

LC-MS/MS

Qin et al.25

Roasted coffee

36


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611

612 613


Table 4. Levels of AA and 5-HMF in 8 categories of heat processed food samples Food groups

n

Potato chip

9

AA (μg/kg)

5-HMF (mg/kg)

Meana

Minimum

Maximum

Mean

Minimum

Maximum

251.0

44.8

633.3

3.9

0.8

9.3

345.5

5.2

0.7

23.5

Biscuit

13

119.8

N.D.b

Bread

6

12.6

10.5

14.4

6.2

1.1

12.2

You-tiao

5

112.2

50.3

161.9

1.2

0.6

2.3

Peanut

4

5.3

N.D.

21.1

1.5

0.3

3.3

Fragrant taro

4

206.4

48.5

425.0

2.8

1.3

6.6

Yueliangba

4

114.6

19.4

200.0

21.3

1.7

56.3

Coffee beverage

5

N.D.

N.D.

N.D.

10.2

1.4

23.2

a For

statistical analysis, N.D. samples were assigned as zero.

b N.D.:

Not detected (lower than the limit of detection).

614

37



615

Graphic for table of contents

616

38


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184x74mm (150 x 150 DPI)


Simultaneous Determination of Acrylamide and 5

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