Exposure Assessment of Acetamide in Milk, Beef, and Coffee Using

Subscriber access provided by READING UNIV

Article

Assessment of Exposures to Acetamide in Milk, Beef, and Coffee Using Xanthydrol Derivatization and GC/MS Ramin Vismeh, Diane Haddad, Janette Moore, Chandra Nielson, Bryan D. Bals, Tim Campbell, Allen Julian, Farzaneh Teymouri, A. Daniel Jones, and Venkataraman Bringi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02229 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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

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

Page 1 of 37

Journal of Agricultural and Food Chemistry

1

Exposure Assessment of Acetamide in Milk, Beef,

2

and Coffee Using Xanthydrol Derivatization and

3

GC/MS

4

Ramin Vismeh†, Diane Haddad†, Janette Moore†, Chandra Nielson†, Bryan Bals†, Tim Campbell†,

5

Allen Julian†, Farzaneh Teymouri†, A. Daniel Jones*‡,§, and Venkataraman Bringi#

6

†

7

8

‡

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, 48824, United States

9

10

§

Department of Chemistry, Michigan State University, East Lansing, Michigan, 48824, United States

11

12 13

14 15

Michigan Biotechnology Institute, Lansing, Michigan, 48910, United States

#

Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, 48824, United States

* Phone: +1-517-432-7126; Fax: +1-517-353-9334; E-mail: [email protected]

1 ACS Paragon Plus Environment


16 17

ABSTRACT

18

Acetamide has been classified as a possible human carcinogen, but uncertainties exist about its

19

levels in foods. This report presents evidence that thermal decomposition of N-acetylated sugars

20

and amino acids in heated gas chromatograph injectors contributes to artifactual acetamide in

21

milk and beef. An alternative GC/MS protocol based on derivatization of acetamide with 9-

22

xanthydrol was optimized and shown to be free of artifactual acetamide formation. The protocol

23

was validated using a surrogate analyte approach based on d3-acetamide, and applied to analyze

24

23 pasteurized whole milk, 44 raw sirloin beef, and raw milk samples from 14 different cows,

25

and yielded levels about 10-fold lower than obtained by direct injection without derivatization.

26

The xanthydrol derivatization procedure detected acetamide in every food sample tested, at 390

27

± 60 ppb in milk, 400 ± 80 ppb in beef, and 39000 ± 9000 ppb in roasted coffee beans.

28 29

Keywords:

analytical artifacts, food safety, method validation, N-acetylated sugars, surrogate

30

analyte validation, thermal degradation, acetamide


Page 2 of 37

Page 3 of 37

31


INTRODUCTION:

32

Acetamide is a simple amide with the chemical formula CH3CONH2 and is formed by the

33

dehydration of ammonium acetate, hydrolysis of acetonitrile, 1, 2 or ammonolysis of acetate esters

34

from plant cell walls. 3, 4 Acetamide has been classified by the International Agency for Research

35

on Cancer (IARC) as a Group 2B possible human carcinogen,5 as feeding trials on rats have

36

shown an increase in liver carcinoma.6,7 The simplicity of the molecule suggests that it may be

37

formed naturally or as a byproduct of other processes. Acetamide has been found in tobacco

38

smoke,8 industrial water9 and in beef and poultry liver.10,11 Two previous studies measured

39

acetamide in milk to determine exposure due to insecticides (thiodicarb and methomyl), and

40

surprisingly found acetamide in the milk of control animals as well.10-12 Furthermore, measured

41

acetamide levels in controls varied greatly between the two tests; in the methomyl it ranged from

42

2 - 8 ppm in milk, while only 0.3 – 0.5 ppm in the thiodicarb study. Both tests used GC to

43

measure the acetamide in milk, but neither report described the method validation in detail.

44

Artifacts may be anticipated during GC analysis of acetamide in foods, and particularly in milk

45

because of the abundance of N-acetylated (or acetamido) moieties on sugars, amino acid

46

metabolites and proteins. Commonly occurring N-acetylated compounds include the

47

monosaccharides N-acetylglucosamine and N-acetylneuraminic acid (commonly known as sialic

48

acid), and acetylated amino acids such as N-acetylcysteine. Furthermore, abundant glycoproteins

49

in milk including κ-casein and lactoferrin also contain N-acetyl sugars.13 A 2001 report14

50

measured N-acetylglucosamine and N-acetylgalactosamine in milk following brief ultra-high

51

temperature (~5 s at ~150 ˚C) pasteurization, and found combined levels of approximately 100

52

ppm. These N-acetylated compounds are potential sources of artifacts in acetamide analysis by

53

releasing acetamide. Further support for this idea comes from a recent NMR investigation of 3 ACS Paragon Plus Environment


54

products released from roasting chicory root, which demonstrated that levels of acetamide “were

55

dramatically increased” upon roasting to levels detectable using NMR spectroscopy.15

56

One may postulate that the presence of acetamide in milk, as well as the discrepancy between

57

the thiodicarb and methomyl studies, arises from the thermal breakdown of endogenous N-

58

acetylated metabolites and glycans into acetamide and the parent sugar molecule during

59

vaporization of milk extracts. 16 . For example, Köll et al.17 found that acetamide was the main

60

volatile degradation product resulting from the thermal decomposition of chitin, a polymer of N-

61

acetylglucosamine. Thermal degradation commenced at 200 ˚C, below the boiling point of

62

acetamide (221 ˚C). These results suggest that in hot (> 200 ˚C) GC injectors, N-acetylated

63

compounds decompose to form acetamide, and thus obscure accurate quantitation of native

64

acetamide.

65 66

Alternatives to hot GC injection have been considered, but can often result in other

67

disadvantages. In liquid chromatography, acetamide has low electrospray ionization efficiency

68

and it is challenging to retain and separate acetamide from a complex mixture using reverse-

69

phase HPLC columns. Elias et al. used HPLC-UV to quantitate acetamide in hydrogeothermal

70

waters.18 Although the reported detection limit for acetamide was low (7.7 ppb), acetamide peaks

71

were broad and showed minimal separation from other analytes in a water matrix, which is

72

considered less complex than food extracts. Furthermore, our initial investigations detected trace

73

levels of acetamide as a contaminant in HPLC-grade acetonitrile, and this finding steered us

74

away from using LC/MS for its analysis. To prevent possible artifact formation from thermal

75

degradation, Diekmann et al.8 used on-column (also known as cool-on-column) injection GC/MS

76

for measuring acetamide and acrylamide in cigarette mainstream smoke. Cool-on-column 4 ACS Paragon Plus Environment

Page 4 of 37

Page 5 of 37


77

injection is not applicable to all samples. In cool-on-column injection, everything in the extracts

78

is introduced into the column, and thus complex matrices have the potential to degrade

79

chromatographic performance. Extracts of food matrices probably need extensive cleanup before

80

injection to minimize irreproducibility and avoid long analysis times.

81

One approach to reduce matrix effects and artifacts in direct GC injection is to derivatize

82

acetamide before injection. In 2013, Cho and Shin19 successfully used derivatization with 9-

83

xanthydrol to measure acetamide in drinking water. 9-Xanthydrol has also been used to

84

derivatize acrylamide20 in processed foods such as potato chips.21,22 9-Xanthydrol reacts with

85

acetamide in aqueous solutions at mildly acidic conditions and forms xanthyl-acetamide, which

86

can be detected and quantitated using hot GC injection.

87

In this manuscript, we assess the suitability of 9-xanthydrol derivatization and direct GC/MS

88

injection to measure acetamide in food matrices, particularly milk and beef. The method was

89

compared to direct GC/MS injection without derivatization. We also performed experiments to

90

evaluate whether this protocol generates artifactual acetamide from N-acetylated compounds.

91

Using this method, we demonstrate the abundance of acetamide in milk and beef products

92

marketed in the United States.

93 94

MATERIALS AND METHODS:

95

Reagents and Stock Solutions

96

Acetamide, d3-acetamide, propionamide, 9-xanthydrol and methanol were purchased from

97

Sigma Aldrich (St. Louis, MO). Ethyl acetate and hexane were purchased from Fisher Chemicals

98

(Fair Lawn, NJ). Sodium chloride and acetone were purchased from Macron Fine Chemicals

99

(Center Valley, PA). Acetone was confirmed by GC/MS to be free of detectable acetamide. 5 ACS Paragon Plus Environment


100 101

Water was prepared using a Millipore MilliQ purification system and hydrochloric acid was purchased from EMD Millipore (Burlington, MA).

102

HCl solution (0.5 M) was prepared in methanol by adding 8.25 mL of HCl stock solution (12.1

103

M) to 191.75 mL methanol in a 200 mL volumetric flask. KOH solution (0.7 M) was prepared by

104

dissolving 0.39 g of KOH in 10 mL water. 9-Xanthydrol solution (5%) was prepared by

105

dissolving 2.5 g of 9-xanthydrol in 50 mL of methanol. Stock solutions of acetamide,

106

propionamide and d3-acetamide were prepared in methanol at 1 g/L by dissolving 10 mg of the

107

chemicals in 10 mL of methanol. Working solutions (10, 50 and 100 ppm) of each chemical were

108

prepared by dilution of 1 g/L stock solution accordingly.

109

Milk, Meat and Coffee Samples

110

Twenty-three pasteurized whole milk and 44 raw sirloin beef samples were purchased from

111

different grocery stores within the State of Michigan, USA. In addition, raw, unprocessed, milk

112

was collected from 14 individual cows, over a period of four weeks. Samples were stored in

113

centrifuge tubes in a freezer (below -20 ˚C) until analysis (within 2-3 d of collection).

114

Coffee samples were purchased from different stores in Michigan and were comprised of 18

115

different varieties of roasted coffee beans, 3 varieties of green (raw) coffee beans and 8 varieties

116

of instant coffee.

117

Preparation of Standards and Calibration Curves

118

Standards for calibration curves were prepared by spiking matrix (milk, coffee and beef) with

119

d3-acetamide as the surrogate analyte and propionamide as the internal standard. The surrogate

120

analyte d3-acetamide was used to account for the variable and non-zero presence of native

121

acetamide in food matrices. Neither d3-acetamide nor propionamide were detected in all

122

unspiked food matrices tested. For milk analysis, standards were prepared by spiking 9.0 mL 6 ACS Paragon Plus Environment

Page 6 of 37

Page 7 of 37


123

milk with 0.0, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µg/mL of d3-acetamide as surrogate analyte and 0.5

124

µg/mL of propionamide as internal standard. The spiked milk samples were processed and

125

derivatized as described below. Linear calibration curves based on equal weighting were then

126

prepared from responses of xanthyl-d3-acetamide (relative peak areas of xanthyl-d3-acetamide to

127

xanthyl-propionamide) on the spiked concentration of d3-acetamide. For beef analyses, standards

128

were prepared by spiking 5 g of raw beef with 0.0, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 ppm of d3-

129

acetamide as surrogate analyte and 0.5 ppm of propionamide as internal standard. The spiked

130

beef samples were processed using the extraction and derivatization protocol.

131

For analysis of coffee samples, standards were prepared by spiking 9.0 mL of aqueous coffee

132

bean extract with 0.0, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µg/mL of d3-acetamide as surrogate analyte

133

and 0.5 µg/mL of propionamide as internal standard. The spiked coffee samples were processed

134

using the extraction and derivatization protocol.

135

Calibration curves were then prepared from dependence of responses of xanthyl-d3-acetamide

136

as the surrogate analyte (relative peak areas of xanthyl-d3-acetamide to xanthyl-propionamide)

137

on the spiked concentration of d3-acetamide.

138

approximately 0.64/ppm for milk, 0.59/ppm for beef, and 0.42/ppm for coffee, respectively.

139

Regression coefficients of the calibration curves were all greater than 0.98.

140

Derivatization Procedure

Slopes for the calibration curves were

141

A 9.0-mL aliquot of milk was spiked with internal standard (100 µL from a 50 ppm stock

142

solution of propionamide) and 900 µL of water was added to each milk sample in 15-mL

143

polypropylene centrifuge tubes. Tubes were centrifuged at 16420 ×g for 10 min and the fat layer

144

on top was removed. A 5-mL volume of the defatted milk was then transferred to a new 15 mL

145

tube and 5 mL of 0.5 M HCl solution was added to precipitate proteins. Tubes were centrifuged 7 ACS Paragon Plus Environment


146

as before, and 5 mL of the supernatant was transferred to new tubes, followed by addition of 200

147

µL of 9-xanthydrol solution in methanol (5%). Tubes were incubated at 40 ˚C for 1.5 h, after

148

which 2 mL of 0.7 M KOH solution, 3 g of NaCl and 3 mL of ethyl acetate were added in turn.

149

Tubes were vortexed and centrifuged, and 1.5 mL of the ethyl acetate phase was transferred to a

150

2 mL microcentrifuge tube. Ethyl acetate was evaporated to dryness using a SpeedVac and

151

analytes were dissolved in 150 µL of ethyl acetate. Microcentrifuge tubes were vortexed and

152

then centrifuged at 20817 ×g for 10 min. A 100 µL aliquot of the supernatant was transferred to

153

vials for GC/MS analysis.

154

Raw beef samples were ground, using a meat grinder, frozen by immersion in liquid nitrogen,

155

and processed using a Ninja blender. Five grams of the homogenized beef were placed in 50 mL

156

polypropylene centrifuge tubes, and 3 mL of water and 11.95 mL of methanol and 50 µL of 50

157

ppm propionamide solution were added to each tube. Samples were then shaken for 2 h at 50 ˚C.

158

After 2 h, tubes were centrifuged at 8045 ×g for 10 min. Supernatants were transferred to new

159

tubes, 15 mL of hexane were added to remove lipids. Tubes were vortexed and centrifuged and

160

hexane layers were removed, then placed in a -80 ˚C freezer overnight for protein precipitation.

161

Next day samples were thawed at room temperature, centrifuged and supernatant was used for

162

the derivatization. The supernatant (1.2 mL), 50 µL of 1 M HCl and 200 µL of 5% 9-xanthydrol

163

solution were then added to 2-mL microcentrifuge tubes and incubated at 40 ˚C for 2.5 h with

164

shaking. Solvents were evaporated from the tubes using a SpeedVac. An 800 µL volume of

165

saturated NaCl in water, 60 µL of 0.7 M KOH, and 800 µL of ethyl acetate were added in turn to

166

each tube. Tubes were vortexed for 3 min using a multitube vortexer. A 500 µL aliquot of the

167

ethyl acetate layer was transferred to a new tube and another 800 µL of ethyl acetate was added

168

to the original tube for a second extraction. This time 700 µL of the ethyl acetate was removed 8 ACS Paragon Plus Environment

Page 8 of 37

Page 9 of 37


169

and added to the initial 500 µL. Ethyl acetate then was evaporated using a SpeedVac system and

170

analytes were dissolved in 100 µL of ethyl acetate. Tubes were centrifuged and 60 µL of the

171

supernatant were transferred to GC/MS vials with glass inserts for GC/MS analysis.

172

Finely ground coffee bean samples (12 grams) were Soxhlet extracted with 250 mL of water

173

for 16 h. The resulting extract was used for the derivatization procedure. Instant coffee samples

174

(four of which were pre-portioned by the manufacturer) ranged from 4.0 - 6.6 grams and were

175

each dissolved in 250 mL of boiling water. The resulting matrix was used for the derivatization

176

procedure. A 9.0-mL aliquot of coffee was spiked with internal standard (100 µL from a 50 ppm

177

stock solution of propionamide) and 900 µL of water was added to each coffee sample in 15-mL

178

polypropylene centrifuge tubes. Tubes were centrifuged at 16420 ×g for 10 min. A 5-mL volume

179

of the coffee supernatant was then transferred to a new 15 mL tube and 5 mL of 0.5 M HCl

180

solution was added. Tubes were centrifuged as before, and 5 mL of the supernatant was

181

transferred to new tubes, followed by addition of 200 µL of 9-xanthydrol solution in methanol

182

(5%). Tubes were incubated at 40 ˚C for 1.5 h, after which 2 mL of 0.7 M KOH solution, 3 g of

183

NaCl and 3 mL of ethyl acetate were added in turn. Tubes were vortexed and centrifuged, and

184

1.5 mL of the ethyl acetate phase was transferred to a 2 mL microcentrifuge tube. Ethyl acetate

185

was evaporated to dryness using a SpeedVac and analytes were dissolved in 150 µL of ethyl

186

acetate. Microcentrifuge tubes were vortexed and then centrifuged at 20817 ×g for 10 min. A

187

100 µL aliquot of the supernatant was transferred to vials for GC/MS analysis.

188

GC/MS Method

189

GC/MS analyses were carried out using a 7890A GC system, equipped with 7683 autosampler,

190

interfaced to a 5973C single quadrupole mass spectrometer (Agilent, Santa Clara, CA). The

191

capillary column used for separation of xanthyl derivatives was a 30 m x 0.25 mm i.d., 0.25 µm 9 ACS Paragon Plus Environment


192

film VF5 with 10 m EZ guard (Agilent, Santa Clara, CA). The capillary column used for

193

quantitation of acetamide (no derivatization) was a 30 m x 0.25 mm i.d., 0.25 µm film DB-WAX

194

(Agilent, Santa Clara, CA). Helium was used as the carrier gas. For the 9-xanthydrol method the

195

flow rate was set at 1.2 mL/min. Injection volume was 1.0 µL using split mode (1:8) at 240 ˚C

196

with the following temperature profile: initial column temperature: 40 ˚C and hold for 2 min;

197

increase to 300 ˚C at 20 ˚C /min and hold for 15 min. The total run time was 40 min per sample.

198

The mass spectrometer was operated in electron ionization (EI) mode at 70 eV. The analytes

199

were detected using selected ion monitoring (SIM) mode for the molecular ions (m/z 239, 242

200

and 253 for xanthyl-acetamide, xanthyl-d3-acetamide and xanthyl-propionamide respectively).

201

The dwell time for each channel was 120 ms. For initial compound characterizations, the mass

202

spectrometer was operated in full scan mode (m/z 30-400).

203

For measurements of acetamide without derivatization, the carrier flow rate was set at 1.5

204

mL/min. Splitless injection of 1.0 µL was made at 240 ˚C with the following column temperature

205

profile: initial column temperature at 50 ˚C and hold for 1 min; increase to 250 ˚C at 15 ˚C /min

206

and hold for 2 min. The total run time was 16.3 min/ sample. The mass spectrometer was

207

operated in electron ionization (EI) mode at 70 eV. The analytes were detected using selected ion

208

monitoring (SIM) of molecular ions m/z 59 and 62 for acetamide and d3-acetamide respectively.

209

For initial compound characterization, the mass spectrometer was operated in full scan mode

210

(m/z 30-400) to generate full EI mass spectra.

211 212

RESULTS AND DISCUSSION

213

Artifactual Acetamide Formation During GC/MS


Page 10 of 37

Page 11 of 37


214

To evaluate artifactual formation of acetamide from N-acetylated compounds, we injected

215

solutions of selected N-acetylated compound standards (at various concentrations) directly into

216

the heated injector (240 ˚C) for GC/MS analysis. Compounds injected were N-

217

acetylglucosamine, sialic acid, N-acetylcysteine and two tri-saccharides; 3-sialyllactose (one of

218

the most abundant sugars in bovine milk and colostrum), and 6-sialyl-N-acetyllactosamine. As

219

anticipated, we detected acetamide in each of the samples, thereby demonstrating artifactual

220

formation of acetamide via thermal degradation. Illustrative data for N-acetylglucosamine are

221

shown in Figure 1. On a molar basis, approximately 15-20% of each precursor was converted to

222

acetamide. Based on these observations, we expect artifactual detection of acetamide when milk

223

or beef extracts are analyzed using direct-injection GC/MS because N-acetylated compounds

224

persist even after sample processing.

225

226

Approaches to Circumvent Artifacts in Acetamide Analysis

227

To minimize artifacts, we tried several approaches that proved to be unsuccessful:

228

We performed cool-on-column injection into the GC/MS to circumvent artifactual formation

229

of acetamide. In this technique, the milk extract was injected directly into the column at 50 ˚C

230

instead of 240 ˚C. Although this technique eliminated the breakdown of N-acetylated compounds

231

in the GC inlet, the method proved impractical because the acetamide peak became broader

232

following multiple injections of milk extract, and components in the milk acetone extract

233

plugged the column after only a few samples. We also evaluated a purge-and-trap technique, in

234

which acetamide is purged from the solution into the vapor phase using helium at up to 70 ˚C.

235

Acetamide was first adsorbed from the vapor phase onto a sorbent, and then thermally desorbed

236

onto the GC column. In principal, this technique could have avoided interference from N11 ACS Paragon Plus Environment


237

acetylated compounds due to their low vapor pressure, but the technique proved impractical due

238

to an unacceptably low purge efficiency (less than 5%) of acetamide.

239

Artifacts may be eliminated if acetamide is separated from other interfering compounds before

240

GC/MS analysis. We tested this approach using phenylboronic acid (PBA) resins to covalently

241

bind to sugars, which could remove N-acetylated sugars from the sample prior to GC/MS

242

injection. However, the high levels of lactose in milk overloaded the resin, making this approach

243

infeasible. A membrane nanofiltration technique to remove sugars was also not successful for the

244

same reason. Charcoal and other sorbents also failed to selectively adsorb the N-acetylated

245

sugars.

246

The most promising method to date exploited derivatizing acetamide using 9-xanthydrol, a

247

derivatizing reagent that was previously used for successful analysis of acrylamide in food

248

matrices21,

249

were less severe than for milk and beef. Our experiments, described below, showed that the 9-

250

xanthydrol derivatization method was successful for analyzing acetamide in milk and beef, two

251

matrices considered likely to present high concentrations of N-acetylated compounds. An

252

overview of the 9-xanthydrol derivatization reaction is shown in Figure 2.

22

and to detect acetamide in environmental water samples21 where matrix effects

253

Excess 9-xanthydrol is added to sample containing acetamide. Derivatization proceeds at 40 ˚C

254

at pH

Exposure Assessment of Acetamide in Milk, Beef, and Coffee Using

Recommend Documents