determination of cyclamate in foods
DESCRIPTION
Determination of Cyclamate in FoodsTRANSCRIPT
FOOD COMPOSITION AND ADDITIVES
Determination of Cyclamate in Foods by UltraperformanceLiquid Chromatography/Tandem Mass Spectrometry
ROBERT SHERIDAN and THOMAS KING
New York State Department of Agriculture and Markets, Food Laboratory, Building 7, State Office Campus, Albany, NY
12235
A highly sensitive and selective method that
requires minimal sample preparation was
developed for the confirmation and quantitation of
cyclamate in a variety of foods by high-performance
liquid chromatography/tandem mass spectrometry
(HPLC/MS/MS). Sample preparation consisted of
homogenization followed by extraction and
dilution of cyclamate with water. HPLC separation
was achieved using a bridged ethyl hybrid C18
high-pressure column with a mobile phase
consisting of 0.15% acetic acid and methanol.
Under electrospray ionization negative conditions,
quantitation was achieved by monitoring the
fragment m/z = 79.7 while also collecting parent
ion m/z = 177.9. Two food matrixes, diet soda and
jelly, were subjected to a validation procedure in
order to evaluate the applicability of the method.
The cyclamate limit of detection for both matrixes
was determined to be 0.050 �g/g with a limit of
quantitation of 0.150 �g/g. The correlation
coefficient of the calibration curves was �0.9998
from 0.0005 to 0.100 �g/mL. The method has been
used for the determination of cyclamate in several
foods and the results are presented.
Sugar substitutes are widely used in the production of
various types of foods. The need for noncaloric
sweeteners for diabetics as well as for individuals
concerned with carbohydrate consumption means that there
will be a great demand for such products. Cyclamate
(N-cyclohexylsulfamate) is a noncaloric sweetener used in
many foods in many countries. It is 30 times sweeter than
sucrose and its sweetening effectiveness increases when it is
used in combination with other artificial sweeteners such as
saccharin (1, 2).
Although cyclamate is allowed for use in food in many
countries, it is not approved for use in food in the United
States because of concerns that it and its metabolite may cause
bladder cancer. Subsequent studies have failed to corroborate
the link to bladder cancer; however, the U.S. cyclamate ban
remains. In recent years an increasing amount of food sold in
the United States is imported from regions of the world where
cyclamate is permitted. In those countries, there are no
regulations regarding the amount of cyclamate allowed in
food, and cyclamate has been detected in foods that typically
require added sweeteners at concentrations as high as
5.08 g/kg. For this reason, a simple, selective, and sensitive
analytical method is needed for detecting cyclamate in foods
over a large range of concentrations.
One of the earliest techniques for cyclamate determination
in foods is high-performance liquid chromatographic (HPLC)
separation followed by UV or conductivity detection.
However, because cyclamate lacks a chromophore necessary
for UV detection, a complex and time-consuming
derivatization procedure is required (3–5). This approach
often involves hazardous reagents or specialized hardware
such as post-column extractors. An alternative detection
scheme for cyclamate detection is the use of an
electrochemical detector in conductivity mode (6). Several
gas chromatographic methods have been reported, but they
too require derivatization of cyclamate prior to analysis (7, 8).
Capillary electrophoresis provides an option where separation
and detection can be performed without derivatization;
however extraction and cleanup steps may be necessary in
order to remove potential interferences (9, 10). Perhaps the
most common technique for cyclamate analysis currently is
flow injection followed by spectrophotometric or
electrochemical detection (11–15). These methods typically
incorporate an in-line derivatization procedure necessary for
analyte detection. When the derivitization method is based
upon the measurement of a reducing agent using the Griess
reaction, the presence of ascorbic acid can interfere with
cyclamate detection (12, 13). Atomic absorption detection can
also be used if the cyclamate is first oxidized and precipitated
with lead nitrate (16). One technique of detecting cyclamate
without derivatization involves the addition of a
chromatographic dye (methyl-red) to the mobile phase
followed by UV-Vis absorbance detection (17). Few
published methods, however, use mass spectrometry (MS) as
a detection scheme. Single-stage MS can provide analyte
structural information, greatly improving method selectivity
over most other strategies (2, 18); however, tandem mass
spectrometry (MS/MS) would provide added selectivity and
sensitivity over single-stage MS.
SHERIDAN & KING: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 5, 2008 1095
Received February 12, 2008. Accepted by SG April 30, 2008.Corresponding author’s e-mail: [email protected]
To our knowledge, no method has been published which
uses LC/MS/MS for the determination of cyclamate in food.
This technique would provide improved selectivity and
confirmation confidence over other methods. We present a
method which uses a minimum of sample preparation
followed by HPLC/MS/MS analysis of cyclamate in several
foods. The selectivity of MS/MS greatly reduces the
possibility of analyte interference with co-extractives, and due
to the inherent sensitivity of this technique, the extracted
sample can be diluted while still providing low detection
limits. Results of an ongoing cyclamate monitoring survey, as
well as validation data, are presented here.
Experimental
Apparatus
(a) LC system.—Acquity HPLC, Waters Acquity binary
solvent manager with sample manager, column manager, and
in-line degasser (Waters, Milford, MA).
(b) Mass spectrometer.—Quattro Premier XE (Waters).
(c) Controlling software.—MassLynx 4.1 (Waters).
(d) LC column and guard column.—Bridged ethyl hybrid
(BEH) C18, 1.7 �m, 1.0 � 100 mm (Waters).
(e) Centrifuge.—Thermo Electron (Milford, MA).
Materials and Reagents
(a) Cyclamate standard.—Sigma (St. Louis, MO). A
1 mg/mL cyclamate stock standard was prepared by
dissolving 0.010 g neat cyclamate standard in 10 mL 50%
deionized water–methanol. All working standards are made in
deionized water without matrix matching. A standard curve
1096 SHERIDAN & KING: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 5, 2008
Table 1. MS conditions for 2 cyclamate transition ions
RT,min
Iontransition, m/z
Conevoltage
Dwelltime, s
Collisionenergy, eV
4.84 Quantitative
ion
177.9 > 79.7 45.00 0.10 25.0
Qualitative
ion
177.9 > 177.9 45.00 0.10 12.0
Figure 1. Cyclamate standard, 10 ng/mL, with both transitions (177.9 > 79.7 and 177.9 > 177.9).
consisting of at least 5 points was generated to quantify each
group of samples. Typically, standards of 0.001, 0.005, 0.010,
0.050, and 0.100 �g/mL (ppm) were used, and all samples
found to contain cyclamate were diluted into this range for
quantitation.
(b) Disposable centrifuge tubes.—50 mL (Fisher
Scientific, Worcester, MA).
(c) Disposable syringe filters.—0.2 �m nylon, 25 mm
diameter (Whatman, Florham Park, NJ).
(d) Deionized water source.—Barnstead Nanopure II
(Dubuque, IA).
(e) Disposable syringes.—3 cc Luer Lock (Kendall,
Mansfield, MA).
(f) Polyethylene bags.—Clear View Bag Co. (Albany, NY).
(g) Methanol.—J.T. Baker (Phillipsburg, NJ); HPLC
RESI-analyzed.
(h) HPLC mobile phase.—A: 0.15% acetic acid;
B: 0.15% acetic acid in methanol.
Instrument Conditions
(a) Column temperature.—30�C.
(b) LC program.—The flow was held at 0.080 mL/min
throughout the 11 min run. The initial mobile phase conditions
were 90% A and held for 1 min; by 6 min the mobile phase
consisted of 2.0% A and was held at this condition until
7.0 min. From 7.0 to 7.5 min, the mobile phase was ramped to
the original conditions where they were held for the remainder
of the 11 min program.
(c) Injection volume.—10 �L.
(d) Sample temperature.—10�C.
(e) Divert valve program.—On from 0 to 1 min and from 8
to 11 min.
(f) MS source temperature.—100�C.
(g) Desolvation temperature.—250�C.
(h) Cone gas flow.—300 L/h.
(i) Desolvation gas flow.—900 L/h.
(j) MS mode.—Multireaction monitoring negative.
(k) Injection mode.—Full loop.
A description of MS acquisition conditions is presented in
Table 1.
Sample Preparation/Extraction
Solid food samples were homogenized in a food processor.
Food types such as jelled fruit and gummy materials were
placed in a plastic bag in a –80�C freezer overnight. The
samples were then removed from the freezer, immediately
placed in a polyethylene bag, and crushed to a particle size as
small as possible. A 1.0 g liquid or well-homogenized food
sample was weighed into a 50 mL disposable centrifuge tube,
and 10 mL distilled deionized water was added. The sample
was mixed on a Vortex mixer for 10 min. It was then
centrifuged for 10 min, and 3 mL of the supernatant was
filtered through a 0.2 �m disposable syringe filter. A 1.0 mL
volume of the filtered supernatant was then added to a 10 mL
volumetric flask and diluted to volume with deionized water.
This final solution was transferred to an autosampler vial for
LC/MS/MS analysis.
SHERIDAN & KING: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 5, 2008 1097
Figure 2. Daughter ions produced from the parent m/z = 177.9 cyclamate.
Results and Discussion
Chromatography
Initially, LC/MS cyclamate analysis was performed using a
standard HPLC system coupled to a single-quadrupole MS.
The column used was an Xterra MS C18, 3.5 �m analytical
column with several flow rates applied. This method
produced a poorly resolved cyclamate peak with large peak
width and an inconsistent retention time. Several sample
matrixes also produced interference for m/z = 178 and 79.7
used to identify cyclamate. For these reasons, an alternative
method was sought.
An improvement in chromatographic resolution,
sensitivity, and selectivity was achieved with the use of
ultraperformance LC/MS/MS. This system consisted of a
BEH C18 column with a particle size of 1.7 �m installed on a
Waters Acquity HPLC with a mobile phase of A = 0.15%
acetic acid and B = 0.15% acetic acid in methanol. With a flow
rate of 0.08 mL/min, the mobile phase gradient began at 90%
aqueous and progressed to 98% organic at 7 min. Under these
conditions, cyclamate was well resolved at 4.8 min and the
retention time was stable. The improvement in selectivity and
sensitivity was due to the use of MS/MS; however, it is not
clear why the higher-pressure LC system gave much better
chromatographic resolution. It is possible that the smaller
particle size, narrower column dimensions, and higher
operating pressure combined to improve cyclamate peak
resolution. Figure 1 shows typical chromatograms of the 2
cyclamate transitions generated from a 0.010 �g/mL
cyclamate standard.
Sample Preparation
Because cyclamate is water-soluble and the analysis
technique is extremely selective, a minimum of preparation is
necessary. The aqueous extract can be easily centrifuged,
filtered, and diluted 200-fold, at which point the co-extracted
matrix does not interfere with the analysis. No difficulties with
emulsions being formed during sample extraction were
observed; however, the method was not tested with high-fat
food. The sensitivity of MS/MS allows for low detection
limits even after a large dilution, and no matrix-induced signal
suppression is evident. For this reason, it is not necessary to
matrix-match standards. No sample preparation difficulties
were encountered using this method with foods as varied as
soda, plum butter, dried fruit, candy, and cake.
Mass Spectrometry
All MS conditions were optimized by a flow injection of
cyclamate solution into the MS source. HPLC mobile phase
was pumped into the MS source through a Tee connector
during the flow of cyclamate solution in order to mimic
analytical run conditions. For this experiment, the mobile
phase was set to the initial conditions of the HPLC program,
which was 10% methanol at a rate of 0.080 mL/min. A10 ppm
cyclamate standard in deionized water was injected into this
flow at 20 �L/min. The MS source conditions were set as
described above and the collision gas flow was turned off. The
MS scan type was set to MS1 in order to collect full-scan data.
In this mode, the collision cell and MS2 are used to pass ions
to the detector. Being an anion, the strongest signal for
cyclamate was seen in negative mode, and with a cone voltage
of 45, the quasimolecular ion m/z = 177.9 was observed. With
the collision gas on and the instrument in daughter-scan mode,
m/z = 177.9 was isolated and fragmented while the collision
energy was varied. As the collision energy was increased, a
1098 SHERIDAN & KING: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 5, 2008
Figure 3. Proposed product ion formation schemefrom cyclamate via electrospray ionization/tandemmass spectrometry.
Table 2. Recovery data from limit of quantitation (LOQ)
method spikes
Spike level
Recovery, %
Diet soda JellyDried
tamarindStrawberries
in syrup
LOQ 1 70.3 81.5 78.2 74.7
LOQ 2 70.2 76.9 73.4 77.4
LOQ 3 72.0 85.4 76.5 74.2
10 � LOQ 1 89.2 75.5 81.0 81.5
10 � LOQ 2 88.6 75.2 86.4 81.1
10 � LOQ 3 90.8 78.6 78.2 83.6
100 � LOQ 1 98.9 84.1 86.4 89.8
100 � LOQ 2 94.9 83.2 85.1 79.0
100 � LOQ 3 94.5 85.4 84.3 77.9
1000 � LOQ 1 99.7 86.1 92.6 93.9
1000 � LOQ 2 100 84.5 92.3 93.7
1000 � LOQ 3 96.1 89.7 91.9 94.0
Mean 88.77 82.18 83.86 83.40
SD 11.46 4.64 6.49 7.54
CV, % 12.9 5.7 7.7 9.0
strong signal at m/z = 79.7 was observed and a further increase
in collision energy resulted in a reduction of m/z = 79.7 signal
strength with no other ions produced (Figure 2). A further
increase in collision energy only resulted in a reduction in
signal abundance without producing novel fragments. The
79.7 ion was the same fragment observed by others using
single-stage MS where it was produced in the ion source (2).
Single-stage MS, however, is susceptible to matrix
interference, whereas MS/MS is much more selective owing
to the isolation of the parent ion prior to fragmentation.
Because only one parent-daughter transition was
generated, we chose to also collect the unfragmented
m/z = 177.9. Ideally, at least 2 daughter fragments should be
produced from a given parent ion in order to satisfy generally
accepted requirements of LC/MS/MS confirmation (19);
however, in some cases this is not possible. While not a
traditional ion ratio measurement, the abundance ratio of
m/z = 177.9 > 79.7 to 177.9 > 177.9 can be used to improve
confirmation confidence over simply observing the presence
of m/z = 177.9 > 79.7. This situation is sometimes unavoidable
when dealing with small molecules. We chose to set
acceptance limits of 10% when comparing quantitation ion
(79.7) to qualifier ion (177.9) for confirming the presence of
cyclamate. Figure 3 represents the proposed fragmentation
scheme for generation of the 79.7 ion. Along with the ion ratio
criteria, the retention time of the analyte in the sample must
match that in the standard within 5% in order for the sample to
be considered confirmed.
One technique for maintaining consistent instrument
response over the course of many injections is through the use
of the flow divert valve. By sending the LC flow to waste for
the initial 1 min, much of the water-soluble co-extracted
potential interference is kept out of the MS source. This
increases the number of sample injections that can be made
before the MS source cone needs to be cleaned, thereby
increasing the number of samples that can be analyzed at
one time.
Method Validation
For the purposes of method validation, the limit of
detection (LOD) was set at 50 ng/g (ppb) for all food types.
SHERIDAN & KING: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 5, 2008 1099
Table 3. Results from pomegranate soda with incurred
cyclamate, extracted and analyzed 10 times
Method repeatability
Run Concn, �g/g
1 198
2 205
3 202
4 221
5 204
6 205
7 202
8 207
9 212
10 223
Mean 208
SD 8.3
CV, % 3.98
Table 4. Commodities found to contain cyclamate
Commodity type No. detected Country of origin
High concentration
found, �g/g
Low concentration
found, �g/g
Plum butter 1 Hungary <0.15 0.103
Pitted cherries 1 Bulgaria 0.2 0.2
Dried prunes 4 China, Taiwan 42499 37500
Mangos in syrup 2 China, Taiwan 439 378
Preserved peach 2 China 3939 2.63
Preserved olive 6 China, Hong Kong 3710 25.9
Preserved plum 15 China, Hong Kong, Taiwan 41253 0.56
Pomegranate soda 1 Multiple countries 241 241
Sweetened kumquat 1 China <0.15 <0.15
Sweet orange 1 China 15300 15300
Grape tomato 1 China 1850 1850
Dried fruit candy 3 China 56300 35500
Dried beans 1 China 664 664
Strawberry cake 1 Taiwan 0.34 0.34
1100 SHERIDAN & KING: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 5, 2008
Figure 4. Transitions for (a) yam jelly containing no cyclamate; (b) 50 ppb cyclamate standard; (c) preserved plumcontaining 1781 ppm cyclamate; and (d) dried plum containing 4390 ppm cyclamate.
This concentration produces a signal from both transitions
well above 10 times noise in all matrixes tested, and it is
unlikely that cyclamate would be added as a sweetener at
levels below this amount. Two sample types, jelly and diet
soda, were spiked at 0.050 �g/g in duplicate and extracted
according to the previously described method. Cyclamate was
confirmed in all LOD spikes with retention time and ion ratio
requirements met.
The limit of quantitation (LOQ) was determined to be
3 times the LOD, or 0.150 �g/g. At this concentration, both
ions produced signals well above 10 times noise and no
interferences were observed. To determine accuracy of
quantitation, 4 food matrixes (diet soda, jelly, dried tamarind,
and strawberries in syrup) were spiked at LOQ (0.15 ppm),
10 � LOQ (1.5 ppm), 100 � LOQ (15 ppm), and 1000 � LOQ
(150 ppm) in triplicate. All spikes were prepared by adding
0.15 mL cyclamate solution at the appropriate level. To obtain
the 1000 � LOQ at 150 ppm, 0.15 mL 1000 �g/mL cyclamate
was added to the 1.0 g blank sample. To obtain a higher
spiking level, cyclamate would have to be added in the pure
form, as 1000 �g/mL was the most concentrated solution
prepared. The spikes were extracted and analyzed in
accordance with the method presented. All recoveries were
between 70 and 100% and the overall average of these
recoveries was 84.6% (Table 2). The standard deviation (SD)
values for the diet soda, jelly, dried tamarind, and strawberries
in syrup were 11.46, 4.64, 6.49, and 7.54, respectively. The
coefficient of variation (CV) values for the diet soda, jelly,
dried tamarind, and strawberries in syrup were 12.9, 5.7, 7.7,
and 9.0%, respectively. The lower percent recovery observed
in some matrixes such as strawberries in syrup was most likely
due to higher ion suppression. Although some matrixes may
cause more suppression than others, leading to lower signal
and reduced recovery, recovery rates were still reasonable and
all confirmation criteria were met.
In order to evaluate the linearity of a typical cyclamate
standard curve, a 5 point curve was generated on 3 different
days, and the curve statistics were calculated. The correlation
coefficient for all curves was �0.9995, which included
standards from 0.001 to 0.1 �g/mL. All samples found to
contain cyclamate were diluted so that the quantitation
response was within the calibration curve range. Instrument
precision was evaluated by injecting one standard repeatedly
19 times, which resulted in a CV of 1.06%. Method
repeatability was demonstrated by extracting and analyzing a
pomegranate soda with incurred cyclamate 10 times. This
study resulted in an SD of 8.3 and a CV of 3.98% (Table 3).
Food Samples
In 2007, a surveillance program was begun in order to
determine the prevalence of cyclamate in foods sold in New
York state. Because cyclamate is not used in domestic food
production, the focus of this program was on imported
products that typically contain added sweeteners. In order to
increase the likelihood of identifying foods containing added
cyclamate, products were sampled for analysis based on the
suspicion that they contained undeclared sweeteners and that
these foods were imported from countries where cyclamate is
commonly used. In those countries, regulation may not
require the explicit labeling of all ingredients including those
that may not be permitted in other countries.
Using this method, cyclamate has been identified in a
number of imported products, including plum butter, dried
fruit, and pomegranate soda from China, Taiwan, Hungary,
and Bulgaria (Table 4). The concentration of cyclamate found
in samples varied greatly from a low of �0.15 �g/g in a
sweetened kumquat imported from China to a high of
56 300 �g/g in dried fruit candy imported from China. All of
the foods that contained �4% cyclamate were dried fruit or
dried fruit candy, which typically tastes very sweet.
Figure 4 illustrates the 2 cyclamate transitions of
m/z = 177.9 � 177.9 and 177.9 � 79.7 for (a) yam jelly
containing no cyclamate; (b) 0.050 �g/mL (ppm) standard of
cyclamate; (c) diluted extract of a preserved plum containing
1781 �g/g cyclamate; and (d) diluted extract of a dried plum
containing 4390 �g/g cyclamate. The ratio of transition area in
the 0.050 ppm standard was 37%, while that of the preserved
plum (c) and dried plum (d) were 38 and 37%, respectively.
Additional commodities that contained cyclamate included
sour cherries, dried prune, jarred peach, jarred olive, plum
butter, and pomegranate soda. None of these food types
presented problems with cyclamate detection even with the
minimal sample preparation used in this method. The wide
range of concentrations found illustrates the need for a large
linear dynamic range for quantitation. We were able to
repeatedly generate a curve from 0.001 to 0.1 �g/mL with a
correlation coefficient of 0.9999 or better, which means fewer
dilutions were necessary to quantify cyclamate.
Conclusions
Amethod for the detection and quantitation of cyclamate in
foods using HPLC/MS/MS has been developed. HPLC
provides good chromatographic resolution for both MS/MS
transitions monitored. The selectivity of MS/MS eliminates
the need for an elaborate sample cleanup procedure, and no
co-extracted interferences have presented problems with
cyclamate detection. This method has been validated and used
in a survey of imported foods suspected of containing
cyclamate. Cyclamate was detected in several food types,
including preserved fruit, soda, and juice. The brief sample
preparation combined with the short run time of the HPLC
method means that many samples can be completed in a day,
giving quantitation and instant confirmation.
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