Quantification of urinary N-acetyl-S-(propionamide)cysteine using an on-line clean-up system coupled with liquid chromatography/tandem mass spectrometry

Article abstract of DOI:10.1002/jms.823

Acrylamide has been reported to be present in high-temperature processed foods and normal processed food intake could lead to significant acrylamide exposure. Acrylamide in vivo can be conjugated with glutathione in the presence of glutathione transferase. This conjugation product is further metabolized and excreted as N-acetyl-S-(propionamide)cysteine (NASPC) in the urine. NASPC could be considered a biomarker for acrylamide exposure. The objective of this study was to develop a highly specific, rapid and sensitive method to quantify urinary NASPC, serving as a biomarker for acrylamide exposure assessment. Isotope-labeled [¹³C₃]NASPC was successfully synthesized and used as an internal standard. This urine mixture was directly analyzed using a newly developed liquid chromatographic/tandem mass spectrometric method coupled with an on-line clean-up system. The detection limit for this method was estimated as <5 μg 1^-1(0.4 pmol) on-column. The method was applied to measure the urinary level of NASPC in 70 apparently health subjects. The results showed that the NASPC urinary level was highly associated with smoking. Smokers had a significantly higher urinary NASPC level (135 ± 88 μg g^-1 creatinine) than non-smokers (76 ± 30 μg g ^-1 creatinine). A highly sensitive and selective LC/MS/MS isotope dilution method was successfully established. With an on-line clean-up system, this system is capable of routine high-throughput analysis and accurate quantitation of NASPC in urine. This could be a useful tool for health surveillance for acrylamide exposure in a population for future study. Copyright

Full text of DOI:10.1002/jms.823

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2005; 40: 511–515
Published online 13 February 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.823
Quantification of urinary N-acetyl-S-
(propionamide)cysteine using an on-line
clean-up system coupled with liquid
chromatography/tandem mass spectrometry
Chien-Ming Li,1 Chiung-Wen Hu2 and Kuen-Yuh Wu1,3∗
1
Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Koahsiung, Taiwan
Department of Public Health, Chung Shan Medical University, Taichung, Taiwan
Department of Risk Management, China Medical University, Taichung, Taiwan
2
3
Received 17 September 2004; Accepted 22 December 2004
Acrylamide has been reported to be present in high-temperature processed foods and normal processed
food intake could lead to significant acrylamide exposure. Acrylamide in vivo can be conjugated with
glutathione in the presence of glutathione transferase. This conjugation product is further metabolized
and excreted as N-acetyl-S-(propionamide)cysteine (NASPC) in the urine. NASPC could be considered
a biomarker for acrylamide exposure. The objective of this study was to develop a highly specific,
rapid and sensitive method to quantify urinary NASPC, serving as a biomarker for acrylamide exposure
assessment. Isotope-labeled [13C3]NASPC was successfully synthesized and used as an internal standard.
This urine mixture was directly analyzed using a newly developed liquid chromatographic/tandem mass
spectrometric method coupled with an on-line clean-up system. The detection limit for this method
was estimated as <5 µg l−1(0.4 pmol) on-column. The method was applied to measure the urinary level
of NASPC in 70 apparently health subjects. The results showed that the NASPC urinary level was
highly associated with smoking. Smokers had a significantly higher urinary NASPC level (135 88 µg g−1
creatinine) than non-smokers (76 30 µg g−1 creatinine). A highly sensitive and selective LC/MS/MS
isotope dilution method was successfully established. With an on-line clean-up system, this system is
capable of routine high-throughput analysis and accurate quantitation of NASPC in urine. This could be
a useful tool for health surveillance for acrylamide exposure in a population for future study. Copyright
 2005 John Wiley & Sons, Ltd.
KEYWORDS: acrylamide; N-acetyl-S-(propionamide)cysteine; biomarker; liquid chromatography/tandem mass
spectrometry
various baked goods and cereals.6,7 Lately, daily dietary
exposures to AA have been of great concern5,8–11 because
AA has been classified as a probable human carcinogen and
neurotoxicant. It has been suggested that AA and its active
metabolite glycidamide are alkylating agents. They contain
an electrophilic group and could be attacked by protein
nucleophilic sites and DNA bases to form protein and DNA
adducts. These adducted bases may lead to mutations if not
repaired prior to cell proliferation.12,13
To assess AA exposure, one of the adducted pro-
teins, N-(2-carbamoylethyl)valine (CEVal), in hemoglobin
has been used as a biomarker to differentiate long-term
exposure to AA among smokers, laboratory workers and
non-smokers.12 AA can be detoxified using glutathione trans-
ferase through conjugation with glutathione, resulting in
the urinary excretion of N-acetyl-S-(propionamide)cysteine
(NASPC).14,15 This metabolite accounts for 67 and 41%
of the total metabolites present in rat and mouse urine,
respectively.14 Therefore, the urinary level of NASPC has
INTRODUCTION
Acrylamide (AA) is an important industrial chemical, whose
polymers have various applications such as in water and
soil treatment, tunnels and dam stabilization as a grouting
agent.1,2 AA is also present in tobacco smoke at levels
of 1.1–2.3 µg per cigarette, probably owing to incomplete
combustion or heating of organic matter.3 Recently, AA
has been detected in baked and fried starchy foods, largely
derived from heat-induced reactions (Maillard reactions)
between the free amino acid asparagines and the carbonyl
group of reducing sugars, such as glucose, during baking and
frying.4–6 High AA contents have been reported in French
fries, potato chips, tortilla chips, bread crust, crispbread and
ŁCorrespondence to: Kuen-Yuh Wu, Division of Environmental
Health and Occupational Medicine, National Health Research
Institutes, 100 Shih-Chuan 1st Road, Koahsiung, Taiwan.
E-mail: kywu@nhri.org.tw
Contract/grant sponsor: National Health Research Institutes,
Taiwan; Contract/grant number: EO-pp-93-07.
Copyright  2005 John Wiley & Sons, Ltd.

512
C.-M. Li, C.-W. Hu and K.-Y. Wu
been used as an alternative biomarker for CEVal to assess
daily exposure to AA.16
NASPC analysis was first performed using high-
performance liquid chromatography (HPLC) with fluores-
cence detection.16 However, this method suffered from
possible interference from the biomatrix and was time con-
suming when large numbers of samples were analyzed. This
method requires derivatization to improve the sensitivity
and avoid matrix effects. Alternatively, liquid chromatog-
raphy/tandem mass spectrometry (LC/MS/MS) has high
selectivity and sensitivity and no derivatization step is
required. With the isotope dilution method, a targeted ana-
lyte could be quantitated reliably and accurately
In order to assess the daily human AA intake from a
variety of foods, an isotope dilution LC/MS/MS method
coupled with an on-line clean-up system was developed in
this research to determine urinary NASPC.
Figure 1. Synthesis of N-acetyl-S-(propionamide)cysteine
(NASPC).
EXPERIMENTAL
Chemicals
N-Acetyl-L-cysteine was purchased from ICN Biomedicals
(Aurora, OH, USA). Acrylamide and 13C3-labeled acrylamide
were obtained from Sigma (St. Louis, MO, USA) and
Cambridge Isotopes (Cambridge, MA, USA), respectively.
Sodium hydroxide and ethyl acetate were purchased from
Merck (Darmstadt, Germany). Hydrochloric acid and formic
acid were supplied by Riedel-de Hae¨n (Seelze, Germany). All
analytical-grade solvents were provided by Tedia (Fairfield,
OH, USA).
Automatic sample clean-up system
The column switching system consisted of a switching
valve (Two-Position Microelectric Actuator, Valco, Houston,
TX, USA) and a BETASIL DIOL cartridge (50 ð 3 mm, i.d.,
particle size 5 µm) (Thermo Electron). The switching valve
function was controlled using PE-SCIEX Analyst control
software. The sample solution (20 µl) was loaded on to the
cartridge using an autosampler (PE Series 200, Perkin-Elmer,
Norfolk, CT, USA) and a quaternary pump (PE Series 200,
Perkin-Elmer) delivered 20 mM ammonium acetate in 95%
acetonitrile at a flow-rate of 1 ml minꢀ1 as the loading and
washing buffer. The cartridge was flushed with loading
buffer for 5 min followed by valve switching to the injection
position to inject the sample into the LC system. At 9 min,
the valve was switched back to the loading position for
equilibration of the cartridge and preparation for the next
analysis.
N-Acetyl-S-(propionamide)cysteine synthesis
NASPC was successfully synthesized in our laboratory from
the reaction of N-acetyl-L-cysteine (2.4 g) with acrylamide
°
(0.35 g) at 45 C in 10 ml of acetone–water (1 : 1) (Fig. 1). This
reaction was maintained under alkaline conditions (2.5 M) by
adding 1 g of NaOH. After 5 h, the solution was neutralized
with hydrochloric acid. NASPC was extracted with ethyl
acetate (4 ð 40 ml). The organic phase was dried using a
rotary evaporator and the residue was dissolved in 95%
acetonitrile. The product was purified and separated using an
HPLC system consisting of a semi-preparative Betasil Diol-
60 column (150 ð 10 mm i.d., particle size 5 µm) (Thermo
Electron, Waltham, MA, USA) and a UV detector set at
210 nm. The mobile phase was 95% acetonitrile delivered
at a flow-rate of 3 ml minꢀ1. NAPSC eluted at 16 min
and was collected and dried using a SpeedVac. NASPC
High-performance liquid chromatography
After automatic sample clean-up, the sample was automat-
ically delivered into a microbore BETASIL DIOL column
(150 ð 2.1 mm i.d., particle size 5 µm) (Thermo Electron).
The gradient mode was used to achieve the desired sample
separation using mobile phase A (95% acetonitrile contain-
ing 0.1% formic acid) and mobile phase B (5% acetonitrile
containing 0.1% formic acid) delivered at a flow-rate of
600 µl minꢀ1. Mobile phase A (100%) was used from 0 to
5.5 min followed by a fast gradient applied to switch to 40%
mobile phase B within 0.5 min. Mobile phase B (40%) was
maintained for 4 min before a quick ramp to 100% mobile
phase B, which was maintained for 1.5 min, then back to
100% mobile phase A, which was maintained for another
15 min.
(1CH3 CONH3CH4COOH5CH2S6CH2 CH2 CONH2) was
characterized by the 1H and 13C NMR chemical shifts using
a Varian (Fort Collins, CO, USA) Mer-Vx-300 spectrometer.
Deuterium oxide and methanol-d4 were used as solvents.
NMR chemical shifts are reported as υ values in parts per
million (ppm). The splitting pattern abbreviations are as
follows: s, singlet; br, broad; m, unresolved multiplet due to
2
7
8
1
the field strength of the instrument. H NMR (D2O), υ 8.37
Electrospray ionization tandem mass spectrometry
(ESI-MS/MS)
An API 3000 triple-quadruple mass spectrometer (Applied
(br, 1H, NH), 4.36 (m, 1H, H-3), 3.04 (m, 1H, H-5a), 2.89 (m,
1H, H-5b), 2.86–2.78 (m, 2H, H-6), 2.55 (m, 2H, H-7), 2.03 (s,
3H, H-1). 13C NMR (CD3OD), υ 177.9 (C-8), 177.2 (C-4), 174.2
(C-2), 55.4 (C-3), 36.2 (C-7), 34.9 (C-5), 28.6 (C-6), 23.1 (C-1).
Biosystems/MDS SCIEX, Concord, ON, Canada) equipped
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 511–515

Quantification of urinary NASPC using LC/MS/MS 513
with a TurboIonSpray source was used. NASPC standard
solution (5 mg lꢀ1) was infused into the mass spectrometer
at 10 µl minꢀ1 to optimize the LC/MS/MS operating param-
eters. A Q1 full-scan NASPC spectrum was conducted first
to obtain [M C H]C and [M ꢀ H]ꢀ ions using the positive and
negative modes, respectively. The [M C H]C and [M ꢀ H]ꢀ
ions were selected to acquire the product ion scans for select-
ing the most distinctive precursor/product NASPC ion pairs.
The mass spectrometer was operated in the negative multiple
reaction monitoring (MRM) mode for NASPC quantitation.
This included m/z 233.0 ! 104.0 for NASPC with the dwell
time set at 150 ms and m/z 236.0 ! 107.0 for [3C13]NASPC
with the dwell time set at 100 ms.
3.0E+06
2.5E+06
2.0E+06
1.5E+06
1.0E+06
5.0E+05
0.0E+00
257.3
235.3
100
150
200
m/z
250
300
(A)
3.0E+06
2.5E+06
2.0E+06
1.5E+06
1.0E+06
5.0E+05
0.0E+00
The spraying needle voltage was set to ꢀ3800 V for the
negative mode. The nebulizer and curtain gas flow-rates
were set at 12. Collision-activated dissociation (CAD) and
turbo gas flow-rate were set at 4 and 8, respectively. The
233.3
250
°
source heater probe temperature was set at 450 C. Data
acquisition and quantitative processing were accomplished
using Analyst software, Version 1.1 (Applied Biosystems).
100
150
200
m/z
300
Urine samples
(B)
Human urine samples were collected from 70 apparently
health male military officers (33 non-smokers and 37
smokers). The smoking status of these subjects was obtained
using a questionnaire. The subjects’ ages ranged from 20 to
46 years with body mass index 17.30–28.40. Military officers
were chosen owing to their similar diet and life style. Urinary
NASPC concentrations were adjusted with creatinine and
expressed in µg gꢀ1 creatinine.
Each urine sample (200 µl) was mixed with 700 µl of 95%
acetonitrile containing 0.1% formic acid and 100 µl of internal
standard ([13C3]NASPC, 100 µg lꢀ1). After centrifugation
at 10 000 r.p.m. for 3 min, sample solution (20 µl) was
injected into the analytical system. No further sample
preparation procedures were needed. The urinary creatinine
was determined as the creatinine–picrate complex by
spectrophotometry (U-2000, Hitachi, Tokyo, Japan) using
a wavelength of 520nm in Kaohsiung Medical Hospital.17
Figure 2. (A) Positive and (B) negative full-scan mass spectra
of NASPC obtained by infusion of 5 µg mlꢀ1 standard solution
into the MS system.
is sodium-adducted NASPC [M C Na]C (Fig. 2(A)). In the
negative mode, the predominant ion is m/z 233, correspond-
ing to [M ꢀ H]ꢀ (Fig. 2(B)). The abundance of the [M C H]C
ion appears approximately equal to that of [M ꢀ H]ꢀ in the
full-scan mode. Product ion scan spectra were obtained by
selecting positive and negative precursor ions to collide with
nitrogen. The fragment ion abundance at m/z 104 operated
in the negative mode was three times higher than that in
the positive mode (Fig. 3). The negative mode will be more
sensitive than the positive mode for analysis of NASPC if
m/z 104 is monitored. Similar mass spectra were obtained
for [13C3]NASPC, except that the corresponding ions were 3
Da greater than those of unlabeled NASPC (data not shown).
The negative MRM mode was applied to monitor product
ions at m/z 104 and 107 for NASPC and [13C3]NASPC frag-
mented from precursor ions at m/z 233 and 236, respectively
(Fig. 4). The mass spectrometer was tuned by direct infu-
sion of 5 mg lꢀ1 NASPC standard solution at a flow-rate of
10 µl minꢀ1 to optimize the ion path voltages.
Statistical methods
Data were analyzed using a statistical package (SPSS,
Statistical Package for the Social Sciences). Statistically
significant differences were determined using p-values
obtained with Student’s t-test.
RESULTS AND DISCUSSION
Precision and accuracy of the isotope dilution
LC/MS/MS method
Characterization of
A linear calibration curve from 10 to 500 µg lꢀ1 was
obtained using known amounts of standards in a blank
urine matrix; each calibration solution contained 100 µl of
100 µg lꢀ1 [13C3]NASPC and yielded an equation for the line
of y D 0.0015x ꢀ 0.0087 ꢀr2 D 0.9997ꢁ, as shown in Fig. 5.
The detection limit was estimated to be 5 µg lꢀ1 (0.4 pmol)
on-column (signal-to-noise ratio D 5). The mean recovery
was calculated as the analyte concentration in the urine
and loading buffer (working solution) divided by analyte
concentration in the loading buffer. A working solution was
prepared in a mixture of urine and loading buffer (95%
N-acetyl-S-(propionamide)cysteine using mass
spectrometry
After NASPC and [13C3]NASPC had been synthesized and
purified, the chemical structure of NASPC was determined
and confirmed by NMR, as described in the Experimental sec-
tion. NASPC and [13C3]NASPC were further characterized
using electrospray ionization tandem mass spectrometry
(ESI-MS/MS). Full-scan spectra were obtained by operating
the ESI-MS/MS system in the positive and negative modes
(Fig. 2) In positive mode, the molecular ion is the proto-
nated NASPC ([M C H]C, m/z 235), and the ion at m/z 257
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 511–515

514
C.-M. Li, C.-W. Hu and K.-Y. Wu
2.5E+05
2.0E+05
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
y = 0.0015x - 0.0087
R2 = 0.9997
OH
O
O
C
H2
C
S
NH2
CH
C
H2
H3C
C
C
O
N
H
1.5E+05
H2
104.0
1.0E+05
5.0E+04
0.0E+00
104.0
130.0
130.0
0
100
200
300
400
500
50
100
150
m/z
200
250
Conc. (ng/mL)
(A)
Figure 5. Calibration curve for quantification of NASPC in
human urine.
2.5E+05
2.0E+05
1.5E+05
1.0E+05
5.0E+04
0.0E+00
104.0
162.0
O-
O
O
H
(n D 6) was found to be 86%.
S
C2
NH2
CH
C
C
H3C
C
C
O
N
H
H2
H2
Relative standard deviation (RSD) is used to express
the precision. It was calculated by dividing the standard
deviation by the mean of the data set. Accuracy is expressed
as the percentage relative error (RE), which is given by the
expression: [ꢀxi ꢀ xtꢁ/xt] ð 100%, where xi is the measured
value and xt is the true value. As for the intra-day variation,
the sample was also prepared in the mixture of urine and
loading buffer at 50 µg lꢀ1 and was analyzed three times,
morning, afternoon and evening, within one day. Two
analyses were conducted at each time point to give a total of
six analyses. Based on the data generated from six replicate
50 µg lꢀ1 NASPC spiked urine analyses, the precision and
accuracy of the method were calculated as 13% and 2%,
respectively.
104.0
162.0
233.0
200
50
100
150
m/z
250
(B)
Figure 3. (A) Positive and (B) negative product ion scan mass
spectra of NASPC obtained by infusion of 5 µg mlꢀ1 standard
solution into the MS system.
sample
clean-up
2.5E+03
elution
8.5
Wu et al.16 established an HPLC/fluorescence detection
method for measuring urinary NASPC. The detection
limit and recovery were reported to be 1 pmol and 94%,
respectively. In comparison with that study, our method
provided a better detection limit. The HPLC/fluorescence
method requires sample purification and derivatization with
o-phthalaldehyde and a total time of 4 h was required
for hydrolysis and derivatization. Such a time-consuming
procedure could limit its application to the analysis of human
samples. With our on-line clean-up system coupled with
LC/MS/MS for NASPC detection in human urine, the total
analysis time is only 12 min without any further hydrolysis
and derivatization.
2.0E+03
1.5E+03
1.0E+03
5.0E+02
0.0E+00
(A) NASPC
0
3
6
9
12
15
Time, min
7.0E+03
6.0E+03
5.0E+03
4.0E+03
3.0E+03
2.0E+03
1.0E+03
0.0E+00
8.5
(B) [3C13]-NASPC
Application to human urine samples
Our method was applied first to investigate the effect of
cigarette smoking on the urinary level of NASPC. Urine
samples were collected from 70 apparently healthy military
officers. The mean urinary levels of NASPC observed
for smokers and non-smokers are plotted in Fig. 6. The
mean urinary level of NASPC for the non-smoker group
was 76 š 30 µg gꢀ1 creatinine and for the smoker group
135 š 88 µg gꢀ1 creatinine. Smokers had a significantly
higher urinary level of NASPC than nonsmokers (p < 0.05).
If we did not use creatinine to adjust the urine samples,
the NASPC concentrations in the urine were 113 š 22
and 159 š 43 µg lꢀ1 in the non-smoker and smoker groups,
respectively. It has been reported that AA is widely present
in cigarettes.3 To our knowledge, our study might be the first
study to show that AA exposure through cigarette smoking
results in an increase in urinary NASPC.
0
3
6
9
12
15
Time, min
Figure 4. Chromatograms of (A) NASPC and (B) [3C13]NASPC
generated from analysis of a urine sample collected from a
non-smoker by using the on-line clean-up LC/MS/MS method.
acetonitrile containing 0.1% formic acid) at a concentration
of 50 µg lꢀ1 and was measured using the on-line clean-up
device followed by LC/MS/MS analysis. The mean recovery
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 511–515

Quantification of urinary NASPC using LC/MS/MS 515
the view and policies of the DEHOM/NHRI, or condemnation or
endorsement and recommendation for use on the issue presented.
250
200
150
100
50
*
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Figure 6. NASPC contents (mean š SD) in urine samples from
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A previous study collected 24 h urine samples from 41
AA-exposed workers at an AA factory and measured the
NASPC urinary levels.16 It was found that the NASPC
urinary concentrations ranged from 1872 to 74 412 µg lꢀ1
.
The urinary levels of NASPC for both the smoker and
non-smoker groups in our study were at least 10 times
lower than those for the exposed workers at the AA
factory. One possible reason could be that the workers at
the AA factory experienced relatively high-level exposure
to AA, resulting in a high level of urinary NASPC.
Another possibility might be that the HPLC/fluorescence
detection method might have overestimated the NASPC
levels in urine, especially as crude urine samples contain
considerable amounts of cross-reacting substances and other
structurally related compounds. It has been suggested that
the HPLC/fluorescence method has low specificity.18
CONCLUSIONS
A highly specific and sensitive isotope dilution LC/MS/MS
method for the determination of urinary NASPC was
successfully established. Using an on-line clean-up system,
this method is capable of routine and high-throughput
analysis for monitoring AA exposure. The isotope dilution
method also provides a high degree of confidence in the
method’s performance. We believe that the proposed method
could be a good tool for assessing AA exposure in future
studies, particularly for assessing low exposures from daily
intakes of high-temperature processed foods.
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Acknowledgements
This research was supported by funds from the National Health
Research Institutes, Taiwan, Grant No. EO-pp-93-07. The scientific
content of this paper has been reviewed and approved for publi-
cation by the Division of Environmental Health and Occupational
Medicine of the National Health Research Institutes. Approval for
publication does not necessarily signify that the content reflects
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 511–515