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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


detection limit.
Acrylamide [CH2 CH(C O)NH2 ] has been detected in high Many chromatographic methods for the detection of acryla-
concentrations in common heated starch-rich foodstuffs and has mide in foods have been reported, such as gas chromatography
attracted considerable public concern. Acrylamide is neurotoxic in (GC) [11–15], gas chromatography mass spectrometry (GC–MS)
rodents [1] and humans [2,3], mutagenic in somatic cells in vitro [4] [16–30], high-performance liquid chromatography (HPLC) [31–40],
and in vivo [5] as well as in germ cells in vivo [6], and carcinogenic high-performance thin-layer chromatography (HPTLC) [41,42], and
in rodents [7–9]. liquid chromatography mass spectrometry (LC–MS) [43–81]. The
It has been classified by the International Agency for Research GC and GC–MS methods are based on reactions with derivatiza-
on Cancer (IARC) as probably carcinogenic to humans (group 2A) tion reagents such as trifluoroacetic anhydride [11,16], xanthydrol
[10]. Potato products, such as French fries and chips, are among the [19], and bromine [22,25,28]. On the other hand, the HPLC, HPTLC,
food items containing the highest amounts of acrylamide. However, and LC–MS methods are optimized for use with 2-mercaptobenzoic
no maximum permitted concentration has yet been established acid [36,53] and dansulfinic acid [41,42].
for acrylamide in processed foods in any country. Establishing a Liquid chromatography–tandem mass spectrometry
maximum permitted concentration for foods through monitoring (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
∗ Corresponding author. Tel.: +82 41 850 8811; fax: +82 41 850 8810. [43–52,55–81]. However, these methods result in low sensi-
E-mail address: hshin@kongju.ac.kr (H.-S. Shin). 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
Fig. 1. The addition reaction of d-cysteine to acrylamide.

All organic solvents used were HPLC grade. Potassium hydrox-


ide, hydrogen chloride, methylene chloride, hexane, d-cysteine 2.4. Calibration and quantitation
(99%), acrylamide (99%), acrylamide-d3 (98%) as an internal
standard, acetic acid, and perfluorooctanoic acid (96%) were The calibration curve for the linearity test was established by
obtained from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile and adding 0.1, 0.5, 1, 5, 10, 50, 100, 250, 500 and 1000 ng of acrylamide
methanol (HPLC grade) were purchased form Honeywell Burdick standard to 2.0 g of samples. The corresponding concentrations
and Jackson (SK Chemicals, South Korea). A stock standard solu- of the standard were 0.05, 0.25, 0.5, 2.5, 5, 25, 50, 125, 250, and
tion of acrylamide was freshly prepared prior to use by dissolving 500 ␮g/kg. The spiked samples were extracted and derivatized with
10.0 mg of acrylamide in 9.9 mL of pure water. A known volume of the method selected in the above reaction procedures, and injected
this solution was sequentially diluted in order to obtain a 1.0 mg/L into the LC system. The peak area of the acrylamide standard was
acrylamide standard solution. This solution was used within 1 h used for the construction of the calibration curve.
of its preparation to prevent loss through evaporation, adsorption,
or polymerization. The pure water used in this study was puri- 2.5. Liquid chromatography–mass spectrometry
fied using a Milli-Q-Reagent-Grade water system (ZD20) and had
a resistivity of >17 M. The food samples were purchased from The liquid chromatography was performed with an Agilent 1200
several local supermarkets. series (Agilent, Palo Alto, CA, USA) equipped with a binary pump,
online vacuum degassing system, and autosampler. The analytes
2.2. Samplings 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
Ten brands each of potato chips and coffee were purchased from a flow rate of 0.2 mL/min was used. The mobile phase A contained
a local supermarket, and French fries were bought from five differ- 10 mM acetic acid and 20 mM perfluorooctanoic acid in water, and
ent local fast food restaurants in South Korea. Each sample was the mobile phase B was acetonitrile. The gradient was as follows:
pulverized and homogenized before sampling. 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-
2.3. Derivatization pounds were eluted within 10 min.
The MS–MS detection was performed using an Agilent 6460
Food samples of 2.0 g were placed into a 10 mL glass-stoppered Series Triple Quadruple instrument (Agilent, Palo Alto, CA). The
test tube, and extracted with 6 mL of methylene chloride. The tube mass spectrometer was operated using electrospray ionization in
was sealed and shaken mechanically for 20 min (Jeio Teck, Korea), the positive ion mode (ESI+). The capillary voltage was set to 4.5 kV.
then centrifuged at 3500 rpm for 5 min. The supernatant was then The source temperature was 120 ◦ C and the desolvation tempera-
placed into another 10 mL test tube and mixed with 3 mL of hex- ture was 350 ◦ C. Nitrogen was used as the desolvation gas (flow
ane, and then the analyte was back-extracted with 0.5 mL of pure 480 L/h) and collision gas at a pressure of 3 × 10−3 mbar. Detec-
water with a controlled pH of 6.5. The solution was then shaken tion was performed in the multiple reaction monitoring (MRM)
for 10 min and centrifuged at 3500 rpm for 5 min. The aqueous mode. Fragment voltage and collision energy were optimized for
layer was then transferred into a 2 mL auto vial after being filtered acrylamide, acrylamide-d3, and their cysteine derivatives (Table 1).
through a 0.20 ␮m syringe filter and d-cysteine was added. The
sample vial was placed in a heating block for derivatization. After 3. Results and discussion
derivatization, 10 ␮L of the solution was injected into the LC system.
In order to study the optimal derivatization conditions, the reac- 3.1. Optimization of derivatization conditions
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 Cysteine was used for the derivatization of aldehydes in our
70 min) in water. Derivatization efficiencies were calculated at var- preceding study [82]. In this study, we proposed its application
ious temperatures (40, 50, 60, 70, 80, and 90 ◦ C) for a reaction time to acrylamide detection through an addition reaction at a dou-
of 50 min and at pH 6.5, and pH levels (5.0, 6.0, 6.5, 7.0, 7.5, 8.0, and ble bond. In weak acidic media, the sulfhydryl group of d-cysteine
9.0) for a reaction time of 50 min at 90 ◦ C. is added at the ␤-site double bond of acrylamide to form 2-
The acidity of each sample was controlled using 1 M HCl or 1 M cysteinyl acrylamide (2-amino-3-(3-amino-3-oxo-propyl) sulfanyl
KOH. Because the addition of acetic, citric, or phosphoric buffer propanoic acid) through the reaction depicted in Fig. 1. Cysteine is
solutions for pH control results in ion-suppression, pH was con- less toxic in water than other derivative reagents reported in the
trolled using HCl or KOH solution. The optimum conditions for literature [36,41,42,53] and found to be reactive with arylamide in
acrylamide derivatization were determined using the area of the weak acidic water matrices in this study. As a result, is thought to
formed derivative. 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 over this time period. No remaining acrylamide was detected after
of the reactants. complete reaction at these conditions.
The formation of derivative was studied over the temperature In order to check for possible loss through the evaporation or
range of 40–90 ◦ C and the reaction period in the range of 10–70 min. polymerization of acrylamide during derivatization, acrylamide in
From the experiment, the optimal reaction temperature and time standard solutions spiked at 5.0, 20, and 100 ␮g/kg was analyzed
was 50 min at 90 ◦ C (Fig. 3). The results indicated that the complete by LC–MS/MS before and after heating for 60 min at 90 ◦ C without
reaction of acrylamide and d-cysteine occurred in approximately the reagent. The acrylamide concentrations obtained by the two
50 min. There was no significant variation in reaction yield noted methods for the same standard solutions coincided within a margin
H.-H. Lim, H.-S. Shin / J. Chromatogr. A 1361 (2014) 117–124 121

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- Fig. 5. Comparison of LC–MS/MS without derivatization with the developed method
tion yield with the first 0.5 mL water provided about 65 ± 4.2% for acrylamide analysis in real samples (n = 25).
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 in potato chips were approximately 0.04 ␮g/kg and 0.14 ␮g/kg,
sensitivity without the concentration step. respectively. In comparison with other methods, the LOQ was far
lower than that obtained with LC–MS and LC–MS/MS detection
3.3. LC–MS/MS optimization [44,45,53,58,67,73,81], as described in Table 2.
Using a least squares fit technique, an examination of the typical
The ESI full scan and tandem mass spectra in the positive mode standard curve was undertaken by computing the regression line
were measured for the derivative. As an objective of this study is of peak area ratios for acrylamide derivative to internal standard
the detection of a very small amount of acrylamide residues in (acrylamide-d3-derivative) on the concentration. The regression
samples. The MRM mode was chosen despite a number of tran- line of the peak area of acrylamide derivative on concentration
sitions that needed to be programmed in order to undertake a using the least-squares fit demonstrated a linear relationship with
multi-compound analysis using the LC–MS/MS. In order to perform y = 17.28x − 0.029 in a concentration range of 0.05–500 ␮g/kg and
the analysis in MRM, the transition ion with the highest abundance r2 = 0.999, where x is the acrylamide concentration (␮g/kg) and y
and mass was chosen as the quantification ion. The ion fragmenta- is the peak area ratio of acrylamide-derivative to acrylamide-d3-
tions were m/z 193.0 ([M+H]+ ), m/z 176.1 ([M+H]+ -OH), m/z 130.1 derivative.
([M+H]+ -NH4 CO2 H), and m/z 104.0 ([M+H]+ -C2 H2 NH4 CO2 H). Accuracy and precision were assessed by determining the recov-
Acrylamide is highly hydrophilic and positively charged at pH ery in samples spiked in potato chips. Intra-day accuracy and
6.5, and consequently separation and detection were improved precision were evaluated using five spiked samples at concentra-
with the use of an ion-pairing reagent. Perfluorooctanoic acid tions of 5.0, 20.0, and 100.0 ␮g/kg for potato chips. Furthermore,
proved to be a good ion-pairing agent for the separation of acryl- inter-day accuracy and precision were determined by their recov-
amide. Superior separation and sensitive detection were obtained ery in spiked samples on five different days. The reproducibility of
using 10 mM acetic acid containing 20 mM perfluorooctanoic acid the assay was very good, as shown in Table 3. The accuracy was
and acetonitrile as an organic modifier. Separation was carried out in a range of 92–104% and precisions of the assay were less than
on ZORBAX SB-C8 column (2.1 mm × 50 mm, 1.8 ␮m particle size). 7%. These results indicate that this method was sufficiently repro-
Fig. 4 presents the LC–MS/MS chromatograms of blank samples ducible to permit reliable analysis of the quantity of acrylamide in
and standard samples spiked with an acrylamide concentration of potato chips.
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. 3.5. Occurrence of acrylamide in foods
Furthermore, the derivatives showed no peak broadening or mem-
ory effects, and did not exhibit adsorption effects in the LC system. To test the applicability of the proposed method to the analysis
The retention time of cysteinyl acrylamide was about 2.28 min. Dis- of real samples, the optimized approach was applied to different
crimination by ion selection was very good. Extraneous peaks were kinds of food. As shown in Table 4, acrylamide was detected in a
not observed in the chromatogram of the samples at the retention concentration range of 0.4–496.0 ␮g/kg in all the samples. Acryla-
times of the derivative. 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
3.4. Verification of method performance 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
The method performance was evaluated by determining the 19.1 ␮g/kg (mean 10.6 ␮g/kg).
detection limit, precision, and accuracy of the method using the The same extracts were analyzed by LC–MS/MS without deriva-
new reagent. tization. Data obtained by the two methods for the same samples
Under optimal conditions, the lowest detection limit (LOD) and coincided within a margin of 5% in the concentration range of
limit of quantification (LOQ) of acrylamide in potato chips were 5.0–496.0 ␮g/kg as shown in Fig. 5. Otherwise, acrylamide was not
determined as the concentrations resulting in a minimum signal- detected in food samples with levels of acrylamide found to be
to-noise ratio of 3 and 10, respectively, and the coefficients of under 2.0 ␮g/kg (LOQ of acrylamide) by LC–MS/MS without deriva-
variation for replicate determinations (n = 7) from samples spiked tization. Agreement between the two methods was excellent in
in potato chips were 15% or less. The LOD and LOQ of acrylamide 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

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