Analytica Chimica Acta 555 (2006) 233–237

Determination of cyclamate in foods by high performance liquidchromatography–electrospray ionization mass spectrometry

Ziqiang Huanga,b,1, Jinyu Mab, Bo Chenb,∗, Ying Zhang1, Shouzhuo Yaoa,b,∗a State Key Laboratory of Chemo/Biological Sensing and Chemometrics, Hunan University, Changsha 410082, PR China

b Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of Education, Chemical Research Institute,Hunan Normal University, Hexi, Erliban, Changsha 410081, PR China

Received 20 July 2005; received in revised form 11 September 2005; accepted 13 September 2005Available online 21 October 2005

Abstract

A high sensitive method for determination of cyclamate in foods by ion-pair high-performance liquid chromatography–electrospray ionizationmass spectrometry was developed and validated. The separation was achieved on a C8 column with 5 mM tris(hydroxymethyl)aminomethaneaqueous solution (pH 4.5, adjusted by acetic acid) as mobile phase with an isocratic mode. The quantification of target compound was completedusing a selected ion recording (SIR) atm/z 178 obtained from ESI-mode. Tiopronin was used as internal standard for the quantification of cyclamate.T g/mL, thel to monitore ply versuso©

K

1

hcesusc

mFmdo

(

ates

com-fer-sitivereal

hro-phy

dsationhighortedarti-

inatehas

ningidelypec-

0d

he correlation coefficient of the calibration curve were better than 0.996, in the range of 50–5000 ng/mL. The limit of detection is 5 nimit of quantification is 20 ng/mL. The inter- and intra-day accuracy, precision were investigated in detail. The method can be usedffectively the content of the artificial sweetener in foods. The method has obvious merits such as high sensitivity, specificity and simther methods reported.2005 Elsevier B.V. All rights reserved.

eywords: Cyclamate; Artificial sweetener; HPLC–ESI MS

. Introduction

Cyclamate is a non-caloric sweetener discovered in 1937. Itas been used widely in low-calorie foods and beverages. Whenyclamate is combined with other low-calorie sweeteners, theynhance each other so that the combinations are sweeter than theum of the individual sweeteners. So, cyclamate has been widelysed as a tabletop sweetener, particularly in combination withaccharin. Now, cyclamate is approved for use in more than 50ountries worldwide.

However, some findings in animals suggested that cyclamateight increase the risk of bladder cancer in humans, the U.S.ood and Drug Administration (FDA) banned the use of cycla-ate in 1969. Although more recent animal studies have failed toemonstrate that cyclamate is a carcinogen or a co-carcinogen,ther issues must be resolved before cyclamate can be approved

∗ Corresponding authors. Tel.: +86 731 8865515; fax: +86 731 8865515.E-mail addresses: dr-chenpo@vip.sina.com (B. Chen), szyao001@sina.com

S. Yao).1 Working in Hunan Entry-Exit Inspection and Quarantine Bureau now.

for commercial use as a food additive in the United St[1].

Because the safety of cyclamate to human is not clearpletely, the restricted content level in foods is different in difent countries. Therefore, it is necessary to develop high senanalytical methods for the determination of cyclamate insamples of diet food products.

For the determination of cyclamate, derivative gas cmatography (GC) and high-performance liquid chromatogra(HPLC) are most widely used[2–14]. However, these methoare often complicated and tedious. Cyclamate requires oxidand derivatization. The sensitivity is not enough for somesensitive detection. A technique based on the filter-suppbilayer lipid membranes for the analysis of mixtures of theficial sweeteners was recently reported in the literature[15]. Thismethod represents an improvement in selectivity to discrimdifferent artificial sweeteners. However, its limit of detectionnot a significantly improvement.

Now, the hyphenated techniques for the chemical screeor analyses of synthetic chemicals in foods have been wapplied. HPLC coupled to electrospray ionization mass s

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.09.030

234 Z. Huang et al. / Analytica Chimica Acta 555 (2006) 233–237

trometry (HPLC–ESI MS) has become widespread in the analy-sis of foods[16]. To our knowledge, the method for determiningcyclamate using HPLC–ESI MS has not been reported up-to-date.

In this paper, a high sensitive ion-pair HPLC–ESI MS methodfor analysis of cyclamate in foods is described. The sensitivity ofthe method was significantly higher than other methods reported.Moreover, this method is also much simpler and reagent-saving,time-saving than others.

2. Experimental

2.1. Materials and apparatus

The standards of sodium cyclamate and tiopronin wereobtained from National Research Center for CRMs (Beijing,China). Samples for examination were purchased from super-market (Changsha, China). Ultra-pure water was prepared usinga Millipore Milli-Q purification system (Millipore, Bedford,MA, USA). Other reagents were of analytical grade, includingacetic acid, tris(hydroxymethyl)aminomethane, triethylamine,dibutylamine. Mobile phases used for HPLC were filtered(0.45�m) and ultrasonically degassed before use.

The HPLC system was a Waters (Milford, MA, USA)Alliance 2695 module. The mass spectrometer was a Micro-mass ZQ 2000 (Manchester, UK) equipped with an ESI probea cquii ).

2

lving1 tan-d L ofw y tha tionsw L forc dardw ork-i oomt nw

2

lesw ch ap )w amp s the /mLo wase eret -n na theH rag

were just filtered off and added internal standard, and injectedinto the HPLC–ESI MS system without further pretreatment.

Because the sensitivity of the method was high, the concentra-tions of cyclamate in final sample solutions should be controlledin the linear range. Specially, when the concentrations of cycla-mate in samples were high, the sample solutions must be diluted.

2.4. HPLC–ESI MS analysis

The separation of the compounds was completed on aspherigel analytical column (Johnson, Dalian, China), whichwas packed with 5�m C8 sillica. The mobile phase consistedof 5 mM tris(hydroxymethyl)aminomethane aqueous solution(pH 4.5, adjusted by acetic acid). The column was washed with100% methanol for 5 min after completion of analysis, and thenequilibrated for 10 min with the mobile phase for the next injec-tion. The flow rate was kept at 1 mL/min with an isocratic modeand the column temperature was maintained at 30◦C. Injec-tion volume was 10�L. The column effluent was split, and only0.2 mL/min portion of the column effluent was delivered into theion source of MS. Electrospray ionization was operated in nega-tive ion mode to generate negative ion of cyclamate. The voltageof capillary, cone, extractor and RF lens was set at 4.5 kV, 50, 5and 0.5 V, respectively. The temperature was maintained at 120and 300◦C for source and desolvation, respectively. The gas flowrate for desolvation and cone was set at 200 and 50 L/h, respec-t of1 singa

2

Cali-b aka ninv li-b sl

vinga t ofq ignale

3

3

cultom tew therew .WH ycla-m ficientw te ins such

nd quadrupole analyzer. The control of system and data ang was performed with Masslynx 3.5 workstation (Waters

.2. Preparation of standards

The stock solution of cyclamate was prepared by disso0.0 mg in 10 mL of water. The stock solution of internal sard tiopronin was prepared by dissolving 1.0 mg in 10 mater. A series of standard solutions were then gained bppropriate dilutions of the above-mentioned stock soluith water to reach concentration range of 50–5000 ng/myclamate. In the standard working solutions, internal stanas also added (60 ng/mL). All of stocking solutions and w

ng solutions were stored in a refrigerator and brought to remperature before use. A 10�L aliquot of the standard solutioas then directly injected on the analytical column.

.3. Preparation of samples

As cyclamate has good solubility in water, all of sampere extracted by water. Low water content samples surepared dried fruit ofAreca catechu L (a typical Chinese foodere mashed after adding knowing quantity water. Mixture sles of solid and liquid such as tanned fruits were mashed antire fruit in syrup. Internal standard was also added (60 ngf final concentration in sample solution). Then the samplextracted in an ultrasonic bath for 20 min. The extracts when centrifuged at 12 000 rpm for 10 min at 4◦C. The superatants were filtered through a 0.45�m nylon membrane. The10�L aliquot of the sample solutions were injected intoPLC–ESI MS system. The liquid samples such as beve

r-

e

s

-e

e

ively. The full scan mass spectrum was acquired at a rangem/z00–220. The quantification of cyclamate was completed uselected ion recording atm/z 178 obtained from ESI-mode.

.5. Linearity, limit of detection, limit of quantification

The concentration range covered was 50–5000 ng/mL.ration curves (y = ax + b) were represented by plotting the perea ratios (y) of cyclamate to internal standard (I.S.) tioproersus the concentrations (x) of the calibration standards. Caration curves were obtained from weighted (1/x2) least-square

inear regression analysis of the data.The limit of detection (LOD) was evaluated as the mass gisignal equal to three times of noise (S/N = 3), the limi

uantification (LOQ) was determined as the mass giving a squal to 10 times of noise (S/N = 10).

. Results and discussion

.1. Mobile phase consideration

Because cyclamate is an anion, its retention is very diffin a reverse phase column such as C8, C18, etc. Although theobile phase was 100% H2O, the retention time of cyclamaas about 3 min, near the dead time of the column, andas no difference among C8, –CN, –NH2 and C18 columnshen separation was completed on a C8 column using 100%

2O as a mobile phase, the linearity of response of SIR of cate standards series was very good, the correlation coefas better than 0.998. However, the recovery of cyclamaamples was very low, specially in tanned fruits samples

Z. Huang et al. / Analytica Chimica Acta 555 (2006) 233–237 235

as Jiaotuo, which is a vegetable and a favorable food in Chinaand Japan. When it is made as tinned food, some sweetenersincluding cyclamate, etc. are always added to improve its taste.About 20�g/mL of cyclamate in sample solution could not bedetected. Maybe, there was a strongly ionized repressive effectto cyclamate because of endogenetic components in samples.

Another separation strategy was using ion pair mobilephase to hold cyclamate on a reverse phase column. Weinvestigated the effect of different ion pair regents suchas tris(hydroxymethyl)aminomethane, triethylamine, dibuty-lamine to retention and sensitivity of cyclamate.

Among three reagents, dibutylamine could give the strongestretention for cyclamate. However, the sensitivity of cyclamatewas the lowest using dibutylamine as the ion pair reagent.The retention of cyclamate in triethylamine modifying mobilephase was slightly stronger than that in tris(hydroxymet-hyl) aminomethane. And the sensitivity in tris(hydroxymethyl)aminomethane was higher than that in triethylamine. Con-sidering about the sensitivity and time-saving, tris(hydroxy-methyl)aminomethane was chosen as the ion pair regent forcyclamate analysis in this paper.

To ion pair HPLC, pH can affect the retention of target com-pound significantly. We investigated the affect of pH on the reten-tion of cyclamate, 5 mM tris(hydroxymethyl) aminomethaneaqueous solution was used as a ion pair mobile phase, and the pHof the buffer was adjusted by acetic acid. In general results, ther easingo , ther harpi nizedr s dis-a than4 e isn hosef atea effectd

oft undt wase henh anew more.W RSDo 5%.T

te andit pec-t ntals -m f thec com-p

anol,a anem amat-

Fig. 1. Chromatograms and ESI mass spectrum of standards: (a) total ion current(TIC), (b) SIR chromatogram, (c) ESI mass spectrum of cyclamate, (d) SIRchromatogram of tiopronin. Note: Concentration of cyclamate standard solutionis 100 ng/mL.

ically. The mobile phase for separation could not contain organicsolvent modifier. After separation completed, the column mustbe washed using 100% methanol to wipe off some endogenouscomponents in samples adsorbed on the column.

Because ion-pair reagents in the mobile can result in contam-ination of the ionization source, we paid attention to long termstability of the method. After 300 injections of different samplesolutions, the responses of analytes were stable (RSD<10%).However, the cone of the ionization source should be cleaned(about after 400 injections) because of deposits formed on pro-longed operation, although it did not affect system performance.

3.2. MS conditions

The MS parameters were optimized attentively by flow injec-tion analysis (FIA). ESI is a soft ionization technique. Cyclamicacid is a fairly strong acid, and at any value above pH 3.0, it is

etention time increases and response decreases with decrf pH of the mobile phase. When pH is smaller than 4.0etention time is longer than 10 min. However, the peak ss broader. From the real sample analyses, the strongly ioepressive effect to cyclamate by endogenetic componentppears when the retention time of cyclamate is longermin. When pH value is higher than 5.0, the retention timear the dead time. So, 4.5 of pH value of mobile phase is c

or the method. At this pH value, the retention time of cyclamnd internal standard is suitable. The ionized repressiveisappears.

In this work, 1, 2, 5, 10, 20, 30 mmol/L concentrationsris(hydroxymethyl) aminomethane were tested. It was fohat when 5 mmol/L tris(hydroxymethyl) aminomethanemployed, the peak is sharp and relatively symmetric. Wigher concentrations of tris(hydroxymethyl) aminomethere used, the resulted peak shape was not improved anyhen lower concentrations of that were used, the peak area

f five consecutive injections for cyclamate was higher thanhe precision of the method get worse.

The chromatograms and ESI mass spectrum of cyclamanternal standard tiopronin are shown inFig. 1. The retentionime of cyclamate and tiopronin is 6.7 and 4.3 min, resively under analytical conditions described in the experimeection. The SIR chromatogram of tiopronin is atm/z 162, quasiolecular ion of the compound. And the peak shape o

omponents is symmetric and sharp. Furthermore, twoounds are separated completely (Fig. 1).

In addition, when some organic solvents such as methcetonitrile were added into tris(hydroxymethyl) aminomethobile phase, the retention time of cyclamate decreased dr

236 Z. Huang et al. / Analytica Chimica Acta 555 (2006) 233–237

Fig. 2. SIR chromatogram of prepared dried fruit ofAreca catechu L: (a) SIRchromatogram of tiopronin, (b) SIR chromatogram of cyclamate.

negatively charged. It gave little fragment ions under 50 V conevoltage, and produced only a few fragment ions under 60 V.Therefore, as described in the previous experimental section,in SIR mode, the cone voltage for cyclamate was set at 50 V.Under this condition, them/z was 178, which just correspondedto cyclamic acid after dissociation of a proton.

3.3. Linearity, limit of detection, limit of quantification

Linearity of cyclamate was obtained over concentration rangefrom 50 to 5000 ng/mL. The correlation coefficient of the cal-ibration curve were better than 0.996. In addition, cyclamateresulted in a quite low LOD and LOQ with SIR detection.According to the USA FDA criteria[17], the analyte responseat the LOQ should be at least five times the response of blanbaseline. With acceptable repeatability, recovery, the LOQ ocyclamate was 5 ng/mL. The analyte response at the limit odetection (LOD) should be reliably differentiated from back-ground noise. The LOD of cyclamate was 1 ng/mL.

3.4. Precision and recovery

The precision of the method obtained by three samples containing a beverage, a tinned sweet Jiaotuo and a prepared driefruit of Areca catechu L was evaluated by the intra- and inter-d nten( 3( aget .I 12.3( aget .

esti-g samp ther low-

est (50 ng/mL), near the middle (800 ng/mL) and the highest(4000 ng/mL). The mean recovery was obtained by the deter-mined concentrations as a percentage of the nominal concentra-tions. With 50, 800 and 4000 ng/mL cyclamate added to extractof tinned sweet Jiaotuo measured to contain 53 ng/mL, the recov-ery was 97.3± 3.3, 100.5± 3.1 and 102.3± 2.6%, respectively(n = 6 each).

3.5. Application

The cyclamate contents of various foods were measured(Fig. 2). The results (mean value± S.D., mg/kg,n = 5 each)are listed as follows: three different tinned orange samplesmeasured 5.1± 0.15, 2.3± 0.08 and 14.3± 0.40, four differ-ent tinned Jiaotuo samples measured 20.7± 0.73, 3.2± 0.13,15.8± 0.39 and 18.2± 0.36, two different tinned mango sam-ples measured 31.1± 1.31 and 4.7± 0.16, five different driedfruit of A. catechu L samples measured 41.7± 0.42, 23.5± 0.54,28.9± 0.66, 11.3± 0.35 and N.D., two different beverage sam-ples measured 12.3± 0.15 and N.D., one ice cream sam-ple measured N.D., one compound artificial sweetener sam-ple measured 5671± 221, one sugar sample measured N.D.,seven different integrated alcoholic beverage samples mea-sured 127.9± 2.81, 253.4± 6.34, 272.2± 8.17, 51.0± 1.84,110.4± 1.21, 67.3± 0.81 and N.D., six different glazed fruitsamples measured 21.0± 0.67, 125.2± 3.13, 241.5± 9.90,3 ej ntoa .

4

atei ra-p beend etec-t ntento mer-i therm

A

nalF ety,C 02,2

R

Ana-

ay assays. Intra-day precision data for the cyclamate comg/kg, %RSD given in parentheses,n = 6 each) were 12.1.0%), 3.2 (3.3%) and 41.7 (2.7%), respectively for beverinned sweet Jiaotuo, and driedA. catechu L fruit, respectivelynter-day precision data for the cyclamate content were1.1%), 3.2 (3.1%) and 41.7 (5.1%), respectively for beverinned sweet Jiaotuo, and driedA. catechu L fruit, respectively

The mean recovery of the target compound was invated by adding standard known concentrations to theles at three different concentrations of cyclamate inange of 50–4000 ng/mL at levels corresponding to the

kff

-d

t

,

,

-

2.1± 0.26, 24.5± 0.39 and 104.2± 2.19, two different applelly samples measured 36.2± 0.83 and N.D., three differerange jelly samples measured N.D., 16.3± 0.19, 34.2± 0.72,ll five different wine samples measured N.D., respectively

. Conclusions

An analytical method for determination of cyclamn foods by ion-pair high-performance liquid chromatoghy/electrospray ionization (ESI) mass spectrometry haseveloped. The optimum experimental parameters of the d

or were chosen. The method can be used to monitor the cof the artificial sweetener in foods. The method has obvious

ts such high sensitivity, specificity and simply, etc. versus oethods reported.

cknowledgement

This work was financially supported by the Natiooundation of Key Technologies for Food Safhina (2003AA2Z3515, 2001BA804A21, 03JJY10001BA804A39).

eferences

[1] http://www.caloriecontrol.org/cyclamat.html,http://cis.nci.nih.gov/fact/319.htm.

[2] J. Saurina, L. Hlabangana, D. Garcia-Milla, S. Hernandez-Cassou,lyst 129 (5) (2004) 468–474.

[3] Q.C. Chen, W.L. Yu, J. Wang, Se Pu. 19 (2001) 105–108.[4] D.P. Nikolelis, S. Pantoulias, Anal. Chem. 73 (2001) 5945–5952.

Z. Huang et al. / Analytica Chimica Acta 555 (2006) 233–237 237

[5] M.C. Yebra-Biurrun, Food Addit. Contam. 17 (2000) 733–738.[6] M.M. Choi, M.Y. Hsu, S.L. Wong, Analyst 125 (2000) 217–220.[7] D. Hernando, J. Saurina, S. Hernandez-Cassou, J. Chromatogr. A 859

(1999) 227–233.[8] A.M.K. Sjoberg, et al., J. Assoc. Off. Anal. Chem. 71 (1988) 1212–1214.[9] J.F. Lawrence, et al., J. Assoc. Off. Anal. Chem. 71 (1988) 934–937.

[10] P. Chen, et al., J. Liq. Chrom. Rel. Technol. 23 (2000) 1961–1971.[11] J.F. Lawrence, et al., Analyst 112 (1987) 879–881.[12] I. Casals, et al., J. Chromatogr. A 750 (1996) 397–402.[13] D.K. Das, et al., J. Chromatogr. 52 (1970) 354–356.

[14] D. Murawski, J. Chromatogr. 546 (1991) 351–367.[15] Dimitrios P. Nikolelis, Spyros Pantoulias, Anal. Chem. 73 (2001)

5945–5952.[16] Richard B. Cole, Electrospray Ionization Mass Spectrometry, Funda-

mentals Instrumentation and Applications, John Wiley and Sons, NewYork, 1997.

[17] Guidance for Industry, Bioanalytical Method Validation, U.S. Depart-ment of Health and Human Services, Food and Drug Administration,Center for Drug Evaluation and Research (CDER), Center for VeterinaryMedicine (CVM), May 2001.