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A new derivatization approach with D-cysteine for the sensitive and simple
analysis of acrylamide in foods by liquid chromatography-tandem mass
spectrometry

Article  in  Journal of Chromatography A · August 2014

DOI: 10.1016/j.chroma.2014.07.094 · Source: PubMed

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 Journal of Chromatography A, 1361 (2014) 117–124

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

A new derivatization approach with d-cysteine for the sensitive and

simple analysis of acrylamide in foods by liquid
chromatography–tandem mass spectrometry
Hyun-Hee Lim a , Ho-Sang Shin b,∗
a
Department of Environmental Science, Kongju National University, Kongju 314-701, Republic of Korea
b
Department of Environmental Education, Kongju National University, Kongju 314-701, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: A liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed in order
Received 26 April 2014 to determine the amount of acrylamide in foods after derivatization with d-cysteine. The sulfhydryl
Received in revised form 29 July 2014 group of d-cysteine was added at the ␤-site double bond of acrylamide to form 2-amino-3-(3-amino-3-
Accepted 30 July 2014
oxo-propyl)sulfanyl-propanoic acid. Deuterated acrylamide (acrylamide-d3) was chosen as the internal
Available online 4 August 2014
standard (IS) for analyzing the food samples. Acrylamide was extracted from 2.0 g of food sample with
6 mL of methylene chloride, and the organic extract was diluted with 3 mL of hexane, and then the ana-
Keywords:
lyte was back-extracted with 0.5 mL of pure water. The derivatization of acrylamide was performed in
Acrylamide
Liquid chromatography–tandem mass the water extract. The best reaction conditions (3.0 mg of d-cysteine, a pH 6.5, a reaction temperature of
spectrometry 90 ◦ C, and a heating time of 50 min) were established by the variation of parameters. The formed deriva-
Foods tive was injected into the LC–MS/MS without further extraction or purification procedures. Separation
d-Cysteine and detection were improved with the use of an ion-pairing reagent of perfluorooctanoic acid. Under
Derivatization the established conditions, the limits of detection and the limits of quantification were 0.04 ␮g/kg and
0.14 ␮g/kg, respectively, and the inter-day relative standard deviation was less than 8% at concentrations
of 20 and 100 ␮g/kg. The method was successfully applied to determine the amount of acrylamide in
potato chips, French fries, and coffee.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction requires a sensitive and simple analytical method with a lower

Acrylamide [CH2 CH(C O)NH2 ] has been detected in high
concentrations in common heated starch-rich foodstuffs and has
attracted considerable public concern. Acrylamide is neurotoxic in
rodents [1] and humans [2,3], mutagenic in somatic cells in vitro [4]
and in vivo [5] as well as in germ cells in vivo [6], and carcinogenic
in rodents [7–9].
It has been classified by the International Agency for Research
on Cancer (IARC) as probably carcinogenic to humans (group 2A)
[10]. Potato products, such as French fries and chips, are among the
food items containing the highest amounts of acrylamide. However,
no maximum permitted concentration has yet been established
for acrylamide in processed foods in any country. Establishing a
maximum permitted concentration for foods through monitoring
∗ Corresponding author. Tel.: +82 41 850 8811; fax: +82 41 850 8810.
E-mail address: hshin@kongju.ac.kr (H.-S. Shin).
detection limit.
Many chromatographic methods for the detection of acryla-
mide in foods have been reported, such as gas chromatography
(GC) [11–15], gas chromatography mass spectrometry (GC–MS)
[16–30], high-performance liquid chromatography (HPLC) [31–40],
high-performance thin-layer chromatography (HPTLC) [41,42], and
liquid chromatography mass spectrometry (LC–MS) [43–81]. The
GC and GC–MS methods are based on reactions with derivatiza-
tion reagents such as trifluoroacetic anhydride [11,16], xanthydrol
[19], and bromine [22,25,28]. On the other hand, the HPLC, HPTLC,
and LC–MS methods are optimized for use with 2-mercaptobenzoic
acid [36,53] and dansulfinic acid [41,42].
Liquid chromatography–tandem mass spectrometry
(LC–MS/MS) is a common technique in the analytical area
and is used sensitively to analyze many types of compounds.
Many LC–MS/MS methods for determining acrylamide in
food have been developed without using derivatization
[43–52,55–81]. However, these methods result in low sensi-
tivity [46,51,55,59,62–64,67,68,77,78] and high interference from

http://dx.doi.org/10.1016/j.chroma.2014.07.094
0021-9673/© 2014 Elsevier B.V. All rights reserved.
118 H.-H. Lim, H.-S. Shin / J. Chromatogr. A 1361 (2014) 117–124
the matrices [51,52,54], and require additional clean-up steps
[49–51,55–57,60–64,66–69,71–80].
The aim of this study is to develop a simple and sensitive acryl-
amide determination method using LC–MS/MS after derivatization
with d-cysteine. Several derivatization parameters were studied in
order to select parameters with high sensitivity and low interfer-
ence in the derivative and to optimize the parameters of LC–MS/MS
for analyzing acrylamide in foods. The new method was applied to
potato chips, French fries and coffee.
2. Experimental
2.1. Reagents
All organic solvents used were HPLC grade. Potassium hydrox-
ide, hydrogen chloride, methylene chloride, hexane, d-cysteine
(99%), acrylamide (99%), acrylamide-d3 (98%) as an internal
standard, acetic acid, and perfluorooctanoic acid (96%) were
obtained from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile and
methanol (HPLC grade) were purchased form Honeywell Burdick
and Jackson (SK Chemicals, South Korea). A stock standard solu-
tion of acrylamide was freshly prepared prior to use by dissolving
10.0 mg of acrylamide in 9.9 mL of pure water. A known volume of
this solution was sequentially diluted in order to obtain a 1.0 mg/L
acrylamide standard solution. This solution was used within 1 h
of its preparation to prevent loss through evaporation, adsorption,
or polymerization. The pure water used in this study was puri-
fied using a Milli-Q-Reagent-Grade water system (ZD20) and had
a resistivity of >17 M. The food samples were purchased from
several local supermarkets.
2.2. Samplings
Ten brands each of potato chips and coffee were purchased from
a local supermarket, and French fries were bought from five differ-
ent local fast food restaurants in South Korea. Each sample was
pulverized and homogenized before sampling.
2.3. Derivatization
Food samples of 2.0 g were placed into a 10 mL glass-stoppered
test tube, and extracted with 6 mL of methylene chloride. The tube
was sealed and shaken mechanically for 20 min (Jeio Teck, Korea),
then centrifuged at 3500 rpm for 5 min. The supernatant was then
placed into another 10 mL test tube and mixed with 3 mL of hex-
ane, and then the analyte was back-extracted with 0.5 mL of pure
water with a controlled pH of 6.5. The solution was then shaken
for 10 min and centrifuged at 3500 rpm for 5 min. The aqueous
layer was then transferred into a 2 mL auto vial after being filtered
through a 0.20 ␮m syringe filter and d-cysteine was added. The
sample vial was placed in a heating block for derivatization. After
derivatization, 10 ␮L of the solution was injected into the LC system.
In order to study the optimal derivatization conditions, the reac-
tion was performed for various amounts of d-cysteine (0.5, 1.0, 2.0,
3.0, 4.0, and 5.0 mg) and reaction times (10, 20, 30, 40, 50, 60, and
70 min) in water. Derivatization efficiencies were calculated at var-
ious temperatures (40, 50, 60, 70, 80, and 90 ◦ C) for a reaction time
of 50 min and at pH 6.5, and pH levels (5.0, 6.0, 6.5, 7.0, 7.5, 8.0, and
9.0) for a reaction time of 50 min at 90 ◦ C.
The acidity of each sample was controlled using 1 M HCl or 1 M
KOH. Because the addition of acetic, citric, or phosphoric buffer
solutions for pH control results in ion-suppression, pH was con-
trolled using HCl or KOH solution. The optimum conditions for
acrylamide derivatization were determined using the area of the
formed derivative.
Fig. 1. The addition reaction of d-cysteine to acrylamide.
2.4. Calibration and quantitation
The calibration curve for the linearity test was established by
adding 0.1, 0.5, 1, 5, 10, 50, 100, 250, 500 and 1000 ng of acrylamide
standard to 2.0 g of samples. The corresponding concentrations
of the standard were 0.05, 0.25, 0.5, 2.5, 5, 25, 50, 125, 250, and
500 ␮g/kg. The spiked samples were extracted and derivatized with
the method selected in the above reaction procedures, and injected
into the LC system. The peak area of the acrylamide standard was
used for the construction of the calibration curve.
2.5. Liquid chromatography–mass spectrometry
The liquid chromatography was performed with an Agilent 1200
series (Agilent, Palo Alto, CA, USA) equipped with a binary pump,
online vacuum degassing system, and autosampler. The analytes
were separated using a 50 mm × 2.1 mm Eclipse Plus C18 column
with a 1.8 ␮m particle size (Agilent, USA). A binary gradient with
a flow rate of 0.2 mL/min was used. The mobile phase A contained
10 mM acetic acid and 20 mM perfluorooctanoic acid in water, and
the mobile phase B was acetonitrile. The gradient was as follows:
at first B = 0%, and then it was increased to 100% after 8 min, before
being decreased back to 0% without any holding time. All com-
pounds were eluted within 10 min.
The MS–MS detection was performed using an Agilent 6460
Series Triple Quadruple instrument (Agilent, Palo Alto, CA). The
mass spectrometer was operated using electrospray ionization in
the positive ion mode (ESI+). The capillary voltage was set to 4.5 kV.
The source temperature was 120 ◦ C and the desolvation tempera-
ture was 350 ◦ C. Nitrogen was used as the desolvation gas (flow
480 L/h) and collision gas at a pressure of 3 × 10−3 mbar. Detec-
tion was performed in the multiple reaction monitoring (MRM)
mode. Fragment voltage and collision energy were optimized for
acrylamide, acrylamide-d3, and their cysteine derivatives (Table 1).
3. Results and discussion
3.1. Optimization of derivatization conditions
Cysteine was used for the derivatization of aldehydes in our
preceding study [82]. In this study, we proposed its application
to acrylamide detection through an addition reaction at a dou-
ble bond. In weak acidic media, the sulfhydryl group of d-cysteine
is added at the ␤-site double bond of acrylamide to form 2-
cysteinyl acrylamide (2-amino-3-(3-amino-3-oxo-propyl) sulfanyl
propanoic acid) through the reaction depicted in Fig. 1. Cysteine is
less toxic in water than other derivative reagents reported in the
literature [36,41,42,53] and found to be reactive with arylamide in
weak acidic water matrices in this study. As a result, is thought to
be a useful derivative reagent for arylamide.
 H.-H. Lim, H.-S. Shin / J. Chromatogr. A 1361 (2014) 117–124 119

Table 1
Ion mode, precursor ion, product ions, fragment voltage, and collision energy of acrylamide, acrylamide-d3, 2-cysteinyl acrylamide and 2-cysteinyl acrylamide-d3 .

Compound Molecular formula Molecular Ion mode Precursor ion Product ions (m/z) Fragment Collision
weight [M+H]+ voltage (V) energy (eV)

Acrylamide C3 H5 ON 71 Positive 72.0 [M+H]+ 72.0 ([M+H]+ ) 20 5

55.0 ([C2 H3 CO]+ ) 20 5
44.0 ([CONH2 ]+ ) 20 10

Acrylamide-d3 C3 H2 D3 ON 74 Positive 75.0 75.0 ([M+H]+ ) 30 3

[M+H]+ 58.1 ([C2 D3 CO]+ ) 30 7
44.1 ([CONH2 ]+ ) 30 9

2-Cysteinyl acrylamide C6 H12 O3 N2 S 192 Positive 193.1 [M+H]+ 193.1 ([M+H]+ ) 70 5

176.1 ([M+H]+ -OH) 70 5
130.1 ([M+H]+ -NH4 CO2 H) 70 10
104.1 ([M+H]+ -C2 H2 NH4 CO2 H) 70 10

2-Cysteinyl acrylamide-d3 C6 H9 D3 O3 N2 S 195 Positive 196.1 [M+H]+ 196.1 ([M+H]+ ) 70 5

179.1 ([M+H]+ -OH) 70 5
133.1 ([M+H]+ -NH4 CO2 H) 70 10
107.1 ([M+H]+ -C2 H2 NH4 CO2 H) 70 10

Fig. 2. The peak area of the derivative as a function of the amount of d-cysteine (A) and solution pH (B) (the reaction was performed for 50 min at 90 ◦ C, n = 3; and with an
ion selection of m/z 193.0).

It is important that the derivative can significantly enhance

ionization efficiency during ESI-MS measurement in the positive
mode. The characteristic ions facilitate the identification and quan-
tification of acrylamide present in a complex matrix. The derivative
was 20 fold more sensitive than acrylamide at the same conditions
using LC–MS/MS, and had good chromatographic peak shape. The
derivative is thought to be sufficiently sensitive for injection by
LC–MS/MS, therefore, the formed derivative was designed to be
directly injected into the LC–MS/MS without an extraction proce-
dure.
In order to obtain optimal conditions for acrylamide derivati-
zation, the effects of the reagent concentration, acidity, reaction
temperature, and time were examined.
The effect of the amount of d-cysteine in concentration ranges
of 0.1–1.0% (w/v) was examined from samples spiked into potato
chip powder. The strongest peak area enhancement was observed
when the amount of added d-cysteine was 3.0 mg in the extract
solution (6.0 mg/mL) at pH 6.5 and a reaction time of 50 min at
90 ◦ C, and was maintained persistently beyond that (Fig. 2A). To
study the effect of acid concentration on the reaction of acrylamide
with d-cysteine, pH was controlled within the range of 5–9. The
Fig. 3. Acrylamide reactivity according to reaction temperature and reaction time
results demonstrated that the maximum peak area was obtained (this experiment was performed at pH 6.5, using 6.0 mg/g d-cysteine, n = 3, and with
at a pH 6.5 (Fig. 2B). At this juncture, we cannot explain why the an ion selection of m/z 193.0).
120 H.-H. Lim, H.-S. Shin / J. Chromatogr. A 1361 (2014) 117–124

Fig. 4. LC–MS/MS chromatogram of blank samples (A), standard samples spiked with an acrylamide concentration of 0.05 ␮g/kg (B) and 1.0 ␮g/kg (C), and real samples (D,
acrylamide in potato chips was quantified as a concentration of 7.5 ␮g/kg).

optimum pH was 6.5 despite this value being higher than the pKa
of the reactants.
The formation of derivative was studied over the temperature
range of 40–90 ◦ C and the reaction period in the range of 10–70 min.
From the experiment, the optimal reaction temperature and time
was 50 min at 90 ◦ C (Fig. 3). The results indicated that the complete
reaction of acrylamide and d-cysteine occurred in approximately
50 min. There was no significant variation in reaction yield noted
over this time period. No remaining acrylamide was detected after
complete reaction at these conditions.
In order to check for possible loss through the evaporation or
polymerization of acrylamide during derivatization, acrylamide in
standard solutions spiked at 5.0, 20, and 100 ␮g/kg was analyzed
by LC–MS/MS before and after heating for 60 min at 90 ◦ C without
the reagent. The acrylamide concentrations obtained by the two
methods for the same standard solutions coincided within a margin
 H.-H. Lim, H.-S. Shin / J. Chromatogr. A 1361 (2014) 117–124
of 3%, therefore, it is thought that the evaporation or polymerization
of acrylamide during derivatization does not take place.
As a result, the results indicate that the optimal reaction con-
ditions for acrylamide with d-cysteine were to use 3.0 mg of
d-cysteine, pH 6.5, and a reaction time of 50 min at 90 ◦ C.
3.2. Optimization of extraction conditions
In previous studies, acrylamide has been extracted from food
samples with water or an organic solvent [40,56]. If water is used
as a solvent, several additional extractions with organic solvents are
needed to reduce the final volume. However, we selected methy-
lene chloride, which has relatively high solubility for acrylamide, as
the first extraction solvent, and then the methylene chloride layer
was mixed with hexane to reduce the solubility of acrylamide in
organic solvent relative to water. Acrylamide in the organic phase
was back-extracted with a small amount of water. The above serial
extraction steps were tested using water of pH 6.5. The extrac-
tion yield with the first 0.5 mL water provided about 65 ± 4.2%
and the continuous twice extraction with 0.5 mL water provided
high extraction efficiencies above 92 ± 2.8% (n = 3). The first extract
only was used for further derivatization in order to obtain better
sensitivity without the concentration step.
3.3. LC–MS/MS optimization
The ESI full scan and tandem mass spectra in the positive mode
were measured for the derivative. As an objective of this study is
the detection of a very small amount of acrylamide residues in
samples. The MRM mode was chosen despite a number of tran-
sitions that needed to be programmed in order to undertake a
multi-compound analysis using the LC–MS/MS. In order to perform
the analysis in MRM, the transition ion with the highest abundance
and mass was chosen as the quantification ion. The ion fragmenta-
tions were m/z 193.0 ([M+H]+ ), m/z 176.1 ([M+H]+ -OH), m/z 130.1
([M+H]+ -NH4 CO2 H), and m/z 104.0 ([M+H]+ -C2 H2 NH4 CO2 H).
Acrylamide is highly hydrophilic and positively charged at pH
6.5, and consequently separation and detection were improved
with the use of an ion-pairing reagent. Perfluorooctanoic acid
proved to be a good ion-pairing agent for the separation of acryl-
amide. Superior separation and sensitive detection were obtained
using 10 mM acetic acid containing 20 mM perfluorooctanoic acid
and acetonitrile as an organic modifier. Separation was carried out
on ZORBAX SB-C8 column (2.1 mm × 50 mm, 1.8 ␮m particle size).
Fig. 4 presents the LC–MS/MS chromatograms of blank samples
and standard samples spiked with an acrylamide concentration of
0.05 ␮g/kg and 1.0 ␮g/kg. For the LC separation of the derivatives,
use of the non-polar stationary phase was found to be efficient.
Furthermore, the derivatives showed no peak broadening or mem-
ory effects, and did not exhibit adsorption effects in the LC system.
The retention time of cysteinyl acrylamide was about 2.28 min. Dis-
crimination by ion selection was very good. Extraneous peaks were
not observed in the chromatogram of the samples at the retention
times of the derivative.
3.4. Verification of method performance
The method performance was evaluated by determining the
detection limit, precision, and accuracy of the method using the
new reagent.
Under optimal conditions, the lowest detection limit (LOD) and
limit of quantification (LOQ) of acrylamide in potato chips were
determined as the concentrations resulting in a minimum signal-
to-noise ratio of 3 and 10, respectively, and the coefficients of
variation for replicate determinations (n = 7) from samples spiked
in potato chips were 15% or less. The LOD and LOQ of acrylamide
121
Fig. 5. Comparison of LC–MS/MS without derivatization with the developed method
for acrylamide analysis in real samples (n = 25).
in potato chips were approximately 0.04 ␮g/kg and 0.14 ␮g/kg,
respectively. In comparison with other methods, the LOQ was far
lower than that obtained with LC–MS and LC–MS/MS detection
[44,45,53,58,67,73,81], as described in Table 2.
Using a least squares fit technique, an examination of the typical
standard curve was undertaken by computing the regression line
of peak area ratios for acrylamide derivative to internal standard
(acrylamide-d3-derivative) on the concentration. The regression
line of the peak area of acrylamide derivative on concentration
using the least-squares fit demonstrated a linear relationship with
y = 17.28x − 0.029 in a concentration range of 0.05–500 ␮g/kg and
r2 = 0.999, where x is the acrylamide concentration (␮g/kg) and y
is the peak area ratio of acrylamide-derivative to acrylamide-d3-
derivative.
Accuracy and precision were assessed by determining the recov-
ery in samples spiked in potato chips. Intra-day accuracy and
precision were evaluated using five spiked samples at concentra-
tions of 5.0, 20.0, and 100.0 ␮g/kg for potato chips. Furthermore,
inter-day accuracy and precision were determined by their recov-
ery in spiked samples on five different days. The reproducibility of
the assay was very good, as shown in Table 3. The accuracy was
in a range of 92–104% and precisions of the assay were less than
7%. These results indicate that this method was sufficiently repro-
ducible to permit reliable analysis of the quantity of acrylamide in
potato chips.
3.5. Occurrence of acrylamide in foods
To test the applicability of the proposed method to the analysis
of real samples, the optimized approach was applied to different
kinds of food. As shown in Table 4, acrylamide was detected in a
concentration range of 0.4–496.0 ␮g/kg in all the samples. Acryla-
mide was detected in the concentration range of 0.4–14.3 ␮g/kg
(mean 5.5 ␮g/kg) in potato chips and in the high concentration
of 77.7–496.0 ␮g/kg (mean 244.0 ␮g/kg) in French fries. The con-
centration of acrylamide in roasted coffee ranged from 6.5 to
19.1 ␮g/kg (mean 10.6 ␮g/kg).
The same extracts were analyzed by LC–MS/MS without deriva-
tization. Data obtained by the two methods for the same samples
coincided within a margin of 5% in the concentration range of
5.0–496.0 ␮g/kg as shown in Fig. 5. Otherwise, acrylamide was not
detected in food samples with levels of acrylamide found to be
under 2.0 ␮g/kg (LOQ of acrylamide) by LC–MS/MS without deriva-
tization. Agreement between the two methods was excellent in
samples with levels of the analyte above 5.0 ␮g/kg. Therefore, it
122 H.-H. Lim, H.-S. Shin / J. Chromatogr. A 1361 (2014) 117–124

Table 2
Comparison of analytical methods for determining acrylamide in various foods.

Reference Matrix Preparation method Derivatization Measurement LOD (␮g/kg) LOQ (␮g/kg)

[44] Fried potatoes SPE – LC–ESI-MS/MS 12 30

[45] Various food products – – LC–MS/MS 0.1 2
[48] Grilled meat, chicken samples – – HPLC–MS 0.5 5.0
[51] Cocoa and coffee LLE–SPE – LC–MS/MS 5.5 9.6
[53,54] Crispbreads, roasting of coffee LLE or SPE 2-Mercaptobenzoic acid LC–MS 4–17 17–30
[58] Crispbread sample SPE – UHPLC–MS/MS 1 3
[67] Coffee, potato chips, biscuits SPE – LC–MS 2 6
[73] Infant rice cereals, cereal-based foods SPE – LC–MS/MS 1 3
[81] Various food products SPE – LC–MS/MS 0.2 0.8
This study Potato chips, coffee, French fries LLE d-Cysteine LC–MS/MS 0.04 0.14

SPE, solid-phase extraction; LLE, liquid–liquid extraction.

Table 3
Intra-day and inter-day laboratory precision and accuracy results for the analysis of acrylamide in various foods (n = 5).

Spiked conc. (␮g/kg) Intraday measured value Interday measured value

Mean ± SD (mg/kg) Accuracy (%) Precision (%) Mean ± SD (mg/kg) Accuracy (%) Precision (%)

5.0 5.1 ± 0.21 101.2 4.1 4.9 ± 0.19 98.4 3.9

20.0 20.2 ± 1.27 100.9 6.3 19.0 ± 0.50 95.0 2.6
100.0 102.1 ± 2.60 102.1 2.5 99.8 ± 3.13 99.8 3.1

Table 4
Analytical results of acrylamide in foods.

Samples Number of samples Analytical results (␮g/kg)

Minimum Maximum Mean ± SD

Potato chip 12 0.4 14.3 5.5 ± 4.5

Roasted coffee 5 6.5 19.1 10.6 ± 5.2
French fry 10 77.7 496.0 244.0 ± 139.4

seems that there is nearly no loss of acrylamide during derivatiza-
tion.
4. Conclusion
A rapid and simple derivatization approach for the sensitive
analysis of acrylamide has been proposed in this study. The qual-
ification of acrylamide can be facilitated by the ions that come
from cysteine during mass spectrometric analysis. Sensitivity can
be enhanced 20 fold by derivatization combined with the usage
of MS/MS. Compared with previous sample pretreatment, the sim-
plified procedure proposed in this paper provides a highly efficient
strategy for the acrylamide analysis of complex food samples. Exist-
ing acrylamide was successfully detected in a sample of only 2 g.
This method offers the easier manipulation of samples, shorter
analysis duration and opportunities for the acrylamide analysis in
the case of small sample sizes.
The developed method confirmed the existence of acrylamide
residues in potato chips, French fries, and coffee, providing an
important tool for evaluating the behavior of acrylamide in food.
Acknowledgment
This work was supported by the South Korean Ministry of
Environment (MOE) as part of “the Environmental Health Action
Program.”
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