Investigations on the degradation of aspartame using high-performance liquid chromatography/tandem mass spectrometry

Article abstract of DOI:10.1016/j.cclet.2014.04.012

Aspartame is a widely used sweetener, the long-term safety of which has been controversial ever since it was accepted for human consumption. It is unstable and can produce some harmful degradation products under certain storage conditions. A high-performa

Full text of DOI:10.1016/j.cclet.2014.04.012

G Model
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Chinese Chemical Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
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Original article
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Investigations on the degradation of aspartame using
high-performance liquid chromatography/tandem mass spectrometry
*
Q1 Jie-Ping Sun, Qiang Han, Xiao-Qiong Zhang, Ming-Yu Ding
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6
Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Department of Chemistry, Tsinghua University, Beijing 100084, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Aspartame is a widely used sweetener, the long-term safety of which has been controversial ever since it
was accepted for human consumption. It is unstable and can produce some harmful degradation
products under certain storage conditions. A high-performance liquid chromatography/tandem mass
spectrometry method was developed for the simultaneous analysis of aspartame and its four
degradation products, including aspartic acid, phenylalanine, aspartyl-phenylalanine and 5-benzyl-3,6-
dioxo-2-piperazieacetic acid in water and in diet soft drinks. Aspartame and its four degradation
products were quantified by a matrix matched external standard calibration curve with excellent
Received 7 February 2014
Received in revised form 20 March 2014
Accepted 9 April 2014
Available online xxx
Keywords:
High-performance liquid chromatography/
tandem mass spectrometry
Aspartame
correlation coefficients. The limits of detection were 0.16–5.8
mg/L, which exhibited higher sensitivity
than common methods. This method was rapid, sensitive, specific and capable of eliminating matrix
interferences. It was also applied to the study of the degradation of aspartame at various pH and
temperatures. The results indicated that aspartame was partly degraded under strong acidic or basic
conditions and the extent of degradation increased with increasing temperature.
ß 2014 Ming-Yu Ding. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Degradation products
Cola
7
8
1. Introduction
(DKP), which was the degradation product of APM, was lacking and 27
needed further relevant research.
28
9
The artificial sweetener, aspartame (
L
-aspartyl-
L
-phenylalanine
While APM is harmless itself, it has poor stability at various pH 29
and temperatures, and thus has different degrees of degradation, 30
which can produce aspartic acid (ASP), phenylalanine (PHE), 31
aspartyl-phenylalanine (ASP-PHE) and DKP [7]. These substances, 32
especially DKP, may be harmful to the metabolic processes in the 33
human body [4,8]. What is worse, the diet soft drinks which 34
contain APM at different pH, depending on the matrix, may also be 35
stored under different temperatures. Therefore, it is important to 36
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methyl ester, APM) has long been commonly used as a substitute of
sugar in low-calorie meals, soft drinks and frozen desserts [1] due
to a degree of sweetness 180 times higher than sucrose [2]. The
Joint Food and Agriculture Organization/World Health Organiza-
tion Expert Committee on Food Additives approved the use of APM
in 1982, with a recommended acceptable daily intake of 0–40 mg/
kg body weight [3]. Nevertheless, the issue of safety of APM has
been controversial since it was discovered [4,5]. A number of
studies claimed some negative health effects from APM ingestion.
It was reported that APM can cause changes in behavior (such as
depression and insomnia), alteration in vision, and mental
retardation, especially in children [2]. Furthermore, APM was also
suspected as carcinogenic [6]. Under this controversy about the
safety of APM, the European Commission required the European
Food Safety Authority (EFSA) to re-evaluate the security of APM in
May 2011. In the assessment process, the expert group of EFSA
found that the data of 5-benzyl-3,6-dioxo-2-piperazieacetic acid
establish
a sensitive, rapid and accurate method for the 37
simultaneously detection of APM and its four degradation 38
products. Furthermore, exploring the degradation of APM at 39
various pH and temperatures is of great significance. A variety of 40
analytical methods, including liquid chromatography [1,8] and 41
capillary zone electrophoresis [9], have been reported for the 42
quantitative analysis of APM, its degradation products and the 43
other sweeteners. However, the sensitivities of these methods are 44
limited and serious matrix interferences can hardly be avoided. In 45
addition, it is difficult to chromatographically separate the peak 46
response of APM and its degradation products, as they have similar 47
structures [10].
48
It has been reported that single quadrupole mass spectrometry 49
(MS) [7,11] was used to quantify APM, its degradation products 50
and the other sweeteners. Compared to MS, tandem mass 51
*
Corresponding author.
E-mail address: dingmy@mail.tsinghua.edu.cn (M.-Y. Ding).
http://dx.doi.org/10.1016/j.cclet.2014.04.012
1001-8417/ß 2014 Ming-Yu Ding. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: J.-P. Sun, et al., Investigations on the degradation of aspartame using high-performance liquid
chromatography/tandem mass spectrometry, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.012

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spectrometry (MS/MS) possesses further sensitivity and stability
[12]. What is more, with the improvement of chromatographic
techniques, high-performance liquid chromatography (HPLC) has
been developed to shorten the analysis time and increase the
resolution, capacity and sensitivity, especially when it is coupled
with MS/MS [13–15]. Recently, many reports were focused on the
application of HPLC–MS/MS in complex samples analysis [16,17].
However, to the best of our knowledge, to date no systematic study
on the degradation of APM by HPLC–MS/MS has been published.
The aim of this paper is to develop a sensitive, rapid and
accurate method using HPLC–MS/MS to simultaneously analyze
APM and its four degradation products ASP, PHE, ASP-PHE and DKP.
This method is also applied to the study of the degradation of APM
at various pH and temperatures, which includes quantifying the
main degradation products and exploring degraded pathways. The
identification characteristics in the mass spectrum of the five
analytes are also included.
2.2. Instrumentations
80
The HPLC–MS/MS instrumentations used in this paper were all
obtained from Shimadzu (Kyoto, Japan). The HPLC was performed
on a LC-20A system. The separation was carried out on the column
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87
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89
90
91
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93
94
95
96
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99
100
VP-ODS (150 mm ꢀ 2 mm, 4.6
mm). Flow rate of the mobile phase
was controlled by a LC-20AD Pump and CBM-20A Controller. The
gradient solvent system consisted of solvent A (0.1% formic acid in
water, v/v) and solvent B (acetonitrile). The content of solvent B in
the mobile phase for the separation of the tested mixture increased
from 10% to 45% in 5 min and was maintained at 45% for 5–7 min.
The total time for one run was 7 min. The injection volume was
5
m
L. The flow rate was set at 0.2 mL minꢁ1 and the oven
temperature was set at 30 8C.
The HPLC was coupled to a Shimadzu 8030 triple quadrupole
mass spectrometer with an electrospray ionization interface.
Nitrogen was supplied as the nebulizing gas and drying gas at the
flow rates of 2.5 L/min and 15 L/min, respectively. The heat block
temperature was set at 400 8C. For MS/MS measurements, the mass
spectrometer was operated in the multiple reaction monitoring
(MRM) mode. Data acquisition was controlled using Shimadzu
LCMS Lab Solution software.
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2. Experimental
2.1. Materials
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The organic reagents were of HPLC-grade and all other
chemicals were of analytical reagent-grade. APM, ASP and ASP-
PHE were purchased from J&K Chemical (Beijing, China). PHE and
DKP were obtained from Sigma–Aldrich Chemistry (St. Louis, USA).
Acetonitrile was purchased from Fisher Scientific (Hampton, USA).
Formic acid was obtained from Fluka Analytical (Buchs,
Switzerland). Ammonia solution was purchased from Xilong
Chemical (Guangdong, China). Plastic bottled cola was obtained
from a local supermarket.
2.3. Standards
101
Aqueous standard solutions of APM, ASP, PHE, ASP-PHE and
DKP were separately prepared by dissolving the standard materials
in distilled water to give a final concentration of 1000 mg/L and
then were stored at 4 8C in a refrigerator. The solutions used in the
parameter optimization experiments were separately prepared
just prior to use by diluting the standard stock solution of each
102
103
104
105
106
107
Table 1
Tandem mass spectrometry parameters of the five analytes.
Analyte
APM
Structure
Retention
Molecular
Quantitative
Qualitative
Quadrupole
Collision
Quadrupole
time (min) weight (g/mol) transition (m/z) transition (m/z) 1 pre-bias (V) energy (V) 3 pre-bias (V)
6.2
294.15
295.15/120.20
295.15/120.20
295.15/180.30
ꢁ15
ꢁ15
ꢁ30
ꢁ15
ꢁ22
ꢁ11
HO
OMe
NH2
O
O
Ph
O
O
N
H
ASP
PHE
2.1
3.3
133.10
165.10
134.10/74.00
166.10/120.05
134.10/74.00
134.10/87.90
ꢁ16
ꢁ16
ꢁ15
ꢁ15
ꢁ13
ꢁ17
HO
NH2
O
OH
166.10/103.10
166.10/120.05
ꢁ12
ꢁ12
ꢁ30
ꢁ15
ꢁ19
ꢁ24
O
OH
Ph
H2N
HO
ASP-PHE
5.1
280.05
281.05/166.25
281.05/166.25
281.05/235.25
ꢁ15
ꢁ15
ꢁ15
ꢁ15
ꢁ10
ꢁ15
OH
O
NH2
O
O
Ph
Ph
O
O
N
H
DKP
6.3
262.10
263.10/91.10
263.10/91.10
263.10/245.15
ꢁ19
ꢁ19
ꢁ35
ꢁ15
ꢁ16
ꢁ15
H
N
HO
O
N
H
Please cite this article in press as: J.-P. Sun, et al., Investigations on the degradation of aspartame using high-performance liquid
chromatography/tandem mass spectrometry, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.012

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analyte with distilled water to a final concentration of 1 mg/L.
Mixed standard solution was prepared just prior to use by diluting
the standard stock solution of each analyte with distilled water to a
final concentration of 1 mg/L.
A series of the calibration standard water solutions were used
for blank matrix curve construction. APM was at the concentration
ranges of 10–1000
the concentration ranges of 10–500
m
g/L while ASP, PHE, ASP-PHE and DKP were at
g/L. Each calibration
m
standard solution was prepared by mixing appropriate volumes
of five prepared stock solutions into the same volumetric flask and
adjusting the final volume with distilled water. Similarly, a series
of the calibration standard cola solutions were used for cola matrix
curve construction. Each calibration standard solution was
prepared by diluting the cola solution by 50 times.
For degradation study at various pH, six standard APM solutions
(pH 2, 4, 6, 7, 8 and 10) were prepared by diluting the standard
stock APM solution with distilled water to a final concentration of
20 mg/L, and adjusting the pH with formic acid or ammonia
solution. Each solution was stored under 30 8C and was diluted to a
concentration of 1 mg Lꢁ1 before being tested. Similarly, three
standard APM solutions at pH 4, kept and 4, 30 and 60 8C, were
prepared for the degradation study at different temperatures.
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2.4. Sample preparation
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The plastic bottled cola A and cola B were degassed in an
ultrasonic bath for 20 min and filtered via 0.2
mm microporous
film. They were diluted 100 times with distilled water before
injecting into the HPLC–MS/MS system with 5
volume.
mL injection
Fig. 1. (a) Total ion chromatogram (TIC) for five analytes; extracted ion
chromatograms of (b) ASP, (c) PHE, (d) ASP-PHE, (e) APM and (f) DKP.
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3. Results and discussion
originated from the ASP were [ASP+H–HCOOH]+ at m/z 88 and 152
[ASP+H–CH3COOH]+ at m/z 74. The ions assigned to PHE were 153
[PHE+H–HCOOH]+ at m/z 120 and [PHE+H–HCOOH–NH3]+ at m/z 154
103. The loss of one HCOOH (46 Da) explained the presence of an 155
ion at m/z 235 from ASP-PHE, while the ion at m/z 166 was 156
[C6H5CH2CH(COOH)NH3]+. Finally, the ion at m/z 245 was the 157
result of a loss of one H2O (18 Da) from DKP and the ion at m/z 91 158
was [C6H5CH2]+ consistent with the cleavage of the branched chain 159
of the ring. In order to simultaneously determine APM, ASP, PHE, 160
ASP-PHE and DKP, a MRM qualitative method was set up. The most 161
abundant product ion in the mass spectra was used for 162
3.1. Optimization of HPLC–MS/MS conditions
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149
150
151
The study of the ionization of the APM, ASP, PHE, ASP-PHE and
DKP was performed by scanning the m/z ranging from 100 to 500 in
both positive and negative modes. In the results, the more
abundant precursor ions of the analytes were their molecular ions
[M+H]+. Therefore, the analytes were detected in the positive ion
mode in remaining work. In order to get the most sensitive product
ion, the fragmentations of each compound were studied by using
product ion scan mode at various collision energies (CE). The
Quadrupole 1 pre-bias and Quadrupole 3 pre-bias were also
optimized by direct injection of individual analyte solutions. The
characteristic product ion and optimal MS/MS parameters for each
analyte are listed in Table 1. The product ion of APM at m/z 180
represented [C6H5CH2CH(COOCH3)NH3]+. Then it lost one
HCOOCH3 (60 Da) to produce ion at m/z 120. The product ions
quantification.
163
Chromatographic parameters, such as the choice of column, 164
mobile phase composition, flow rate and column temperature, 165
were also tested to obtain the best separation of the five analytes. 166
Our study indicated that 0.2 mL minꢁ1 and 30 8C were the optimal 167
Table 2
Linear relationships and sensitivities for detection of five analytes.
Analyte
APM
Linear range
g/L)
Linear equation
R2
RSD (%) (n = 5)
Limit of detection
g/L)
Limit of quantification
(mg/L)
(
m
(
m
10–1000
10–500
10–500
10–500
10–500
A
B
y = 14,861x + 91,215
y = 15,945x ꢁ 89,588
0.9964
0.9987
2.2
9.7
3.2
5.8
10.6
19.3
ASP
A
B
y = 6683x ꢁ 14,725
y = 6628x + 39,838
0.9999
0.9998
3.4
2.3
2.9
7.7
9.6
18.9
PHE
A
B
y = 21,254x + 94,612
y = 18,807x + 205,795
0.9995
0.9988
1.1
0.16
0.35
0.52
17.4
1.2
ASP-PHE
DKP
A
B
y = 3901x + 294
0.9999
1.0000
1.8
0.73
0.79
2.4
2.6
y = 3742x ꢁ 1127
13.2
A
B
y = 3034x ꢁ 8057
y = 2945x ꢁ 3150
0.9999
0.9999
2.5
2.8
3.1
9.3
13.9
10.3
A – in water solution; B – in cola solution.
Please cite this article in press as: J.-P. Sun, et al., Investigations on the degradation of aspartame using high-performance liquid
chromatography/tandem mass spectrometry, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.012

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J.-P. Sun et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
Table 3
the range of 0.35–5.8
m
g/L and LOQs were in the range of 1.2–
196
197
198
199
Recoveries of five analytes and concentrations of five analytes in cola A.
19.3
mg/L. The LODs and LOQs of the proposed method were of
much higher sensitivity than the reported HPLC method [9]. The
RSDs were less than 18.9%.
Analyte
Spiked
g/L)
R (%)
RSD (%)
Cola A
(
m
(recovery)
(n = 3)
Concentration
RSD (%)
(m
g/L)
(n = 3)
3.3. Analysis of real samples
200
APM
ASP
100
500
102.5
96.6
4.1
9.2
96,250
–
9.3
The validated method was applied to the simultaneous analysis
of APM, ASP, PHE, ASP-PHE and DKP in cola A and cola B. No
obvious signal was detected in cola B. However, the peaks of APM,
ASP-PHE and DKP were detected in cola A without ASP and PHE.
The real concentrations of APM, ASP-PHE and DKP are listed in
Table 3. The results indicated that the APM added in cola A had
partly degraded and the main products were ASP-PHE and DKP. In
addition, to evaluate the accuracy of the method, recoveries were
201
202
203
204
205
206
207
208
209
210
211
212
213
214
100
500
92.6
99.8
3.1
4.8
18.7
11.4
7.8
PHE
100
500
112.5
99.7
2.1
7.3
–
ASP-PHE
DKP
100
500
111.5
99.9
3.5
9.1
11,730
1330
100
500
106.2
100.0
3.5
7.3
10.2
tested. The samples were spiked with 100
analytes and 500 g/L analyte mixture, respectively. The recover-
ies were 96.6–100% and RSDs were less than 9.2% for the 500 g/L
mg/L of the mixture of
m
‘‘n = 300 means separated analysis of three different samples.
m
analyte mixture, which indicated that the method was reliable and
could be used for the determination of the five analytes in real
samples.
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169
170
171
172
173
174
175
176
177
flow rate and oven temperature regarding resolution and total run
time. Under optimal conditions, the total ion chromatogram for the
mixture of the five analytes is shown in Fig. 1(a), extracted ion
chromatograms of the five analytes are shown in Fig. 1 (b)–(f).
The two peaks in Fig. 1(d) represented ASP-PHE and its isomer
[7,18]. The sum of areas of these two peaks was used to quantify
ASP-PHE [18,19]. Because of similar structures, APM and DKP
did not achieve baseline separation. However, with the MRM
qualitative method, these two analytes could still be accurately
quantified.
3.4. Comparison of methods
215
A comparative study of our developed method to other reported
methods in terms of LOD and run time was performed, and the
results are presented in Table 4. It can be seen that the LOD of this
work was far lower than the reported methods, confirming its high
sensitivity. This sensitive method made it possible to detect the
slightly degradation products from APM in beverages and study
their further properties. On the other hand, the run time of this work
was shorter than, or nearly equal to the conventional methods,
which would increase efficiency in detection of real samples. More
importantly, the proposed method could eliminate any matrix
interference and produce exact quantitative results even if the
analytes did not reach baseline separation. This advantage provided
a foundation to discover new degradation products of APM and
degradation products having similar structures. In summary, the
developed method is a sensitive, rapid and accurate method that can
be used for the simultaneous analysis of APM and its four
degradation products, as well as its degradation study.
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224
225
226
227
228
229
230
231
232
178
3.2. Method validation
179
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185
186
187
188
189
190
191
192
193
194
195
The linearity, sensitivity, as well as precision and accuracy of
the proposed method were validated by a series of experiments
described below. Linearity was studied by analyzing mixed water
standard solutions of the five analytes and also mixed cola
standard solution of the five analytes at several concentration
ranges, respectively.
The results of linear regression analysis are shown in Table 2.
Linear regression analysis of blank matrix curve showed that the
correlation coefficients of all the standards were higher than
0.9964. Limits of detection (LODs), which were defined as the
concentrations at three times the signal intensity to noise, were in
3.5. Degradation study of APM under various pH
233
the range of 0.16–3.2
as the concentrations at 10 times the signal intensity to noise, were
in the range of 0.56–10.6 g/L. The relative standard deviations
(RSDs) were less than 3.4%. Similarly, the linear regression analysis
of the cola matrix curve showed that the correlation coefficients of
all the standards were higher than 0.9987. Results for LOD were in
m
g/L. Limits of quantitation (LOQs), defined
Since diet soft drinks which contained APM are at different pH,
depending on their matrixes, and usually stored at different room
temperatures, a degradation study of APM at various pH and 30 8C
was carried out. The 20 mg/L standard APM solutions (pH 2, 4, 6, 7,
8 and 10) were kept at 30 8C. The pH of each solution was checked
every day and formic acid or ammonia solution was carefully
234
235
236
237
238
239
m
Table 4
Comparison of the HPLC–MS/MS method developed in the present work with some reported analytical procedures.
Analytea
Sample
Techniqueb
Limit of detection
0.16–5.8 g/L
Run time (min)
Reference
APM, ASP, PHE, ASP-PHE, DKP
APM, CYC, SAC, ACS-K
Cola
HPLC–MS/MS
CZE–UV
m
7
6
Present work
Beverages
0.50–12.0 mg/L
<15 mg/L
[9]
APM, ACS-K, ALI, CYC, DUL, NEO, NHDC, SAC, SUC
APM, DKP
Beverages, canned fruits and yoghurts
Diet soft drinks
HPLC–ELSD
HPLC–DAD
2-D HPLC
HPLC–ESI-MS
ESI-MS
23
7
[20]
[8]
2–5 mg/L
APM,
L
-ASP,
D-ASP,
L
-PHE,
D
-PHE
Coca-cola
0.16–1.3 mg/L
<0.1 mg/L
70
25
5
[1]
APM, SAC, ACS-K, NHDC, SUC, CYC, ALI, STV
APM, ASP, PHE, ASP-PHE, DKP, PME, MeOH
Beverages, candied fruits and cakes
Water
[11]
[7]
0.1–0.5 mg/L
a
CYC, cyclamate; SAC, saccharin; ACS-K, acesulfame-K; ALI, alitame; DUL, dulcin; NEO, neotame; NHDC, neohesperidin dihydrochalcone; SUC, sucralose; STV, stevioside;
PME, phenylalanine methyl ester; MeOH, methanol.
b
CZE-UV, capillary zone electrophoresis coupled with ultraviolet detection; HPLC-ELSD, high performance liquid chromatography coupled with evaporative light
scattering detector; HPLC-DAD, high performance liquid chromatography coupled with diode-array detector; 2-D HPLC, two-dimensional high-performance liquid
chromatography coupled with on-line post column derivation fluorescence detection and ultraviolet detection; HPLC–ESI-MS, high performance liquid chromatography
coupled with electrospray ionization mass spectrometric detection; ESI-MS, electrospray ionization mass spectrometry.
Please cite this article in press as: J.-P. Sun, et al., Investigations on the degradation of aspartame using high-performance liquid
chromatography/tandem mass spectrometry, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.012

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Fig. 2. (a) Relative amounts of APM found in solutions at pH 2, 4, 6, 7, 8 and 10 at 30 8C after 5 days and 10 days to original APM solution; (b) relative amounts of APM found in
solutions at 4, 30 and 60 8C and pH 4 after 8 days to original APM solution; (c) intensity of degradation products ASP, PHE, ASP-PHE and DKP in solutions at pH 2 (60 8C), pH 10
(60 8C) and 60 8C (pH 4) after 5 days.
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
added to maintain the study pH, if needed (usually none, or only a
negligible volume of the acid or base solution was needed). The
solutions were diluted to a concentration of 1 mg/L for analysis at
the appropriate time every day.
ASP-PHE and DKP. Both ASP and PHE only existed under an acid 257
environment, which indicated the acidic environment was 258
conducive to breaking the –NH– bond of APM. Fig. 3(c) showed 259
the degradation pathways to form ASP and PHE. This result was in 260
The result in Fig. 2(a) showed the remaining APM concentration
ratios to original APM solution at various pH after 5 days and 10
days, respectively, which indicated that APM in strong acid and
base environment was unstable. When the pH approximated 4,
APM degraded more slowly than at other pH. Besides, at room
temperature, APM in the solutions of acidic pH decomposed much
more slowly than in solutions of basic pH. The difference between
the results of 5 and 10 days indicated the extent of degradation of
APM increased over time. The result in Fig. 2(c) showed that the
main product under a strong basic environment was ASP-PHE and
DKP, due to the OHꢁ ion promoting cleavage of –CH2– from APM to
form ASP-PHE and to remove one molecule of methanol to form a
ring. Fig. 3(a) and (b) showed the degradation pathways to form
agreement with previously reported work [7].
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3.6. Degradation study of APM under various temperatures
262
Based on the experiment in Section 3.6, APM degraded more 263
slowly at pH 4. Thus, three sets of 20 mg/L APM solutions at pH 4 264
were separately kept at 4, 30 and 60 8C for degradation. The 265
solutions were diluted to a concentration of 1 mg/L before testing. 266
Three solutions were quantitated every day for APM, ASP, PHE, 267
ASP-PHE and DKP by the developed HPLC–MS/MS method. The 268
result in Fig. 2(b) showed the ratios of remaining APM concentra- 269
tion to original APM solution at 4, 30 and 60 8C after 8 days, which 270
indicated the extent of degradation of APM increased with 271
Fig. 3. Degradation pathways of APM to form (a) ASP-PHE, (b) DKP, (c) ASP and PHE.
Please cite this article in press as: J.-P. Sun, et al., Investigations on the degradation of aspartame using high-performance liquid
chromatography/tandem mass spectrometry, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.012

G Model
CCLET 2956 1–6
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temperature. The degradation of APM at 30 8C showed the APM
was unstable at normal temperatures. This result was in
agreement with previous work in Section 3.4 carried out in
analyzing real samples of cola A. The APM added to cola A had
partly degraded to ASP-PHE and DKP under normal conditions. The
result in Fig. 2(c) showed the main product at 60 8C was DKP.
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4. Conclusion
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A reliable HPLC–MS/MS method for the simultaneous analysis
of APM and its four degradation products ASP, PHE, ASP-PHE and
DKP was developed. The method was based on the simultaneous
monitoring of all ion pairs of the analytes in MRM mode at the
optimal conditions. Under these conditions, there was no
interfering fragment of any monitored analyte by other analytes
also being monitored. Therefore, it could provide accurate
quantitative results even the analytes could not be baseline
separated. The identification characteristics of the mass spectrum
of five analytes were also included. This method could be used to
analyze real samples with satisfactory results.
The study on degradation of APM in solution at various pH
levels and temperatures was carried out easily using this method
to simultaneously determine the relative abundance of APM, ASP,
PHE, ASP-PHE and DKP. Application of this method to the study on
degradation of APM at various pH showed that APM degraded
much more easily in strong acidic and basic solutions, with DKP
and ASP-PHE as major products in basic solutions. Higher
temperatures will promote the degradation of APM, with DKP as
the major product. Based on this work, it indicated the cola
containing APM should be stored under proper conditions. Based
on this work, it is also possible to study the further properties of
APM and its degradation products.
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Acknowledgment
303 Q2
This project was supported by the National Nature Science
Foundation of China (No. 21075074).
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References
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Please cite this article in press as: J.-P. Sun, et al., Investigations on the degradation of aspartame using high-performance liquid
chromatography/tandem mass spectrometry, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.04.012