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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

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Journal of Agricultural and Food Chemistry

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Exposure Assessment of Acetamide in Milk, Beef,

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and Coffee Using Xanthydrol Derivatization and

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GC/MS

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

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

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†

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‡

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

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§

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

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

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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]

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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Keywords:

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

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analyte validation, thermal degradation, acetamide


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INTRODUCTION:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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acetamide.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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injection to minimize irreproducibility and avoid long analysis times.

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

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

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

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

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

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can be detected and quantitated using hot GC injection.

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

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

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

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

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

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marketed in the United States.

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MATERIALS AND METHODS:

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Reagents and Stock Solutions

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

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

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

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


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

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

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

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

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

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

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

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

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Milk, Meat and Coffee Samples

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

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

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

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

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

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

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of instant coffee.

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Preparation of Standards and Calibration Curves

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

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

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

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

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

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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

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

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

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

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

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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-

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

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beef samples were processed using the extraction and derivatization protocol.

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

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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

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

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using the extraction and derivatization protocol.

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

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

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on the spiked concentration of d3-acetamide.

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

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

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Derivatization Procedure

Slopes for the calibration curves were

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

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

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

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

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


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

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

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

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

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

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

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

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vials for GC/MS analysis.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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GC/MS Method

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

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

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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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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RESULTS AND DISCUSSION

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Artifactual Acetamide Formation During GC/MS


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

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

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

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

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

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

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

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

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

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

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persist even after sample processing.

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Approaches to Circumvent Artifacts in Acetamide Analysis

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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sugars.

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

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

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matrices21,

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

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

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

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

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and to detect acetamide in environmental water samples21 where matrix effects

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

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at pH

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