Simultaneous determination of some artificial sweeteners in ternary formulations by FT-IR and EI-MS Nicoleta Tosa, Zaharie Moldovan, and Ioan Bratu Citation: AIP Conf. Proc. 1425, 98 (2012); doi: 10.1063/1.3681976 View online: http://dx.doi.org/10.1063/1.3681976 View Table of Contents: http://proceedings.aip.org/dbt/dbt.jsp?KEY=APCPCS&Volume=1425&Issue=1 Published by the American Institute of Physics.
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Simultaneous Determination Of Some Artificial Sweeteners In Ternary Formulations By FT-IR And EI-MS Nicoleta Tosa*, Zaharie Moldovan* and Ioan Bratu National Institute for Research and Development of Isotopic and Molecular Technologies, 65-103 Donath, 400293 Cluj-Napoca, Romania * Corresponding authors:
[email protected] ,
[email protected] Abstract. Artificial sweeteners are widely used in food, beverage and pharmaceutical industries all over the world. In this study some non-nutritive sweeteners such as aspartame, acesulfame–K, sodium cyclamate and sodium saccharin were simultaneously determined in ternary mixtures using FT-IR and EI-MS measurements. FT-IR method is based on direct measurements of the peak height values and area centered on 1736 cm-1, 836 cm-1, 2854 cm-1 and 1050 cm-1 for aspartame, acesulfame–K, sodium cyclamate and sodium saccharin, respectively. Mass spectrometry determinations show the characteristic peaks at m/z 91 and 262 for aspartame, m/z 43 and 163 acesulfame-K, m/z 83 and 97 for sodium cyclamate and m/z 104 and 183 for sodium saccharin. The results obtained by EI-MS in different formulations are in agreement with the FT-IR ones and provide also essential data concerning the purity grade of the components. It is concluded that FT-IR and EI-MS procedures developed in this work represent a fast, sensitive and low cost alternative in the quality control of such sweeteners in different ternary formulations.
Keywords: non-nutritive sweeteners, FT-IR, EI-MS, ternary formulations. PACS: 33.15.Ta, 33.20Ea, 33.20Tp, 61.66.Hq, 82.80Gk, 82.80.Ms quantitative determination. Thus, determination of aspartame and acesulfame-K has been developed by FT-IR in chloroform/methanol solution [12], whereas aspartame determination in soft drinks was accomplished by attenuated total reflectance FT-IR [13]. Also, quantitative detection by ESI-MS operated in negative ionization mode was used in HPLC analysis of sweeteners in soil and waste water [14]. In this work we extended the quantitative study for mixtures containing more than two components and report on a rapid, sensitive and non-toxic procedure for determination of some sweeteners in different ternary formulations by using FT-IR (KBr pellet technique) and EI-MS methods. These results represent primary steps for applications in food and beverages quality control.
INTRODUCTION Several non-nutritive sweeteners such as sodium cyclamate (SCY), acesulfame-K (ACK), aspartame (ASP) and sodium saccharin (SSA) are used at large scale as a sugar substitute in beverages and food due to their sweet taste and remarkable sweetening power relative to a standard concentration of sucrose. Their sweetness ranges from 30 to 300 times sweeter than sucrose [1] but are also accompanied by a bitter metallic taste-off. Therefore, blends of structurally different sweeteners in binary or ternary formulations mask the aftertaste and produce synergistic sweetening effects [2]. Taking into account the side effects produced by these compounds among human body and environment it is required to be analyzed in order to determine their concentration and purity grade. Highperformance liquid chromatography (HPLC) is the most commonly used method, based either on isocratic reverse-phase (RP) chromatographic separation and ultraviolet (UV) absorbance detection [3], or on ion chromatography (IC) with UV [4] and electrochemical detection [5]. Flow injection [6] as well as cyclic voltammetry [7] methods has been also used for the sweeteners determination. Although FT-Raman [8] and FT-IR spectroscopy is often used for characterization of artificial sweeteners in complexing [9], spectroscopic [10] and voltammetric [11] studies, there are very few application of these methods for
EXPERIMENTAL In this work the artificial sweeteners acesulfame-K (C4H4KNO4S), aspartame (C14H18N2O3), sodium cyclamate (C6H12NNaO3) and sodium saccharine (C7H4NNaO3S·2H2O were purchased from Worton Enterprise Ldt. China, and used as received. They have been considered as references in order to establish the percentage composition of two unknown samples denoted formulation I and formulation II, respectively. Formulation I, containing ACK, ASP and SCY, as well as formulation II, containing ACK, ASP and SSA, were analyzed as received.
Processes in Isotopes and Molecules (PIM 2011) AIP Conf. Proc. 1425, 98-101 (2012); doi: 10.1063/1.3681976 © 2012 American Institute of Physics 978-0-7354-1005-3/$30.00
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FT-IR measurements were performed in the range 4000 to 350 cm-1 on a JASCO 6100 spectrometer (single beam) with a 4 cm-1 resolution, and 700 scans were performed to collect each spectrum. KBr pellet technique for measurements in absorbance mode was employed. Mass spectral data were recorded under 70 eV electron impact on a double focusing mass spectrometer VARIAN MAT-311 set at 100 µA electrons current, with the ion source temperature 150 o C. The inlet system temperature (maintained at the evaporation temperatures appropriate for each compound) was programmed in the range 25-300ºC at an increasing rate of 10ºC/min. Accurate measurements of the fragment ions masses were performed with high resolution (R = 5000, 10 % valley), in the peak matching mode, with PFK ions for reference masses.
deformation of the CH trisubstituted vinyl linked into the heterocycle, at 836 cm-1, that does not overlap with other specific bands of the investigated sweeteners. The ASP spectrum displays at 1736 cm-1 a strong absorption band typical to the stretching vibration of an ester carbonyl group. The strong intensity band at 1665 cm-1 corresponds to the carbonyl stretching vibration (Amide I band), whilst the band at 1548 cm-1 is assigned to the coupling of the NH bending and C-N stretching vibrations (Amide II band) of the secondary amide function. The shoulder at 1585 cm-1 is assigned to C-C stretching of benzene ring [1*]. Also, the peaks in the region 1200-1400 cm-1 might be attributed to the coupled of single-bond C-C and C-O stretching vibration involved in O=C-O groups. The peak at 699 cm-1 corresponds to the C-H out-of-plane deformation specific to an aromatic ring [15]. Sodium cyclamate spectrum (Figure 1) shows for imino group a sharp stretching vibration (υ NH) band at 3279 cm-1 and a strong intensity deformation band (δ NH) at 1447 cm-1. SO2 symmetric stretching vibration is located at 1170 cm-1, whereas the asymmetric one appears at 1220 cm-1 and 1243 cm-1. The CH2 groups of cyclohexene display intense symmetric and asymmetric stretching vibration bands at 2854 cm-1 and 2927 cm-1, respectively. Figure 2 shows the FT-IR spectra of ACK, ASP, SSA and of unknown Formulation II samples with corresponding amounts of 0.74 mg, 0.73 mg, 0.75 mg and 1.08 mg, respectively, in100 mg KBr.
RESULTS AND DISCUSSION FT-IR Studies Figure 1 shows the FT-IR spectra of ACK, ASP, SCY and of unknown Formulation I samples with corresponding amounts of 0.74 mg, 0.73 mg, 0.75 mg and 0.75 mg, respectively, in 100 mg KBr.
FIGURE 1. FTIR spectra of ACK, ASP, SCY and of Formulation I, recorded in KBr matrix. FIGURE 2. FTIR spectra of ACK, ASP, SSA and of Formulation II, recorded in KBr matrix.
The spectrum of acesulfame-K (Figure 1) reveals a strong band at 1657 cm-1, corresponding to the C=O stretching vibration of the amide group (Amide I band), and a very intense band at 1596 cm-1 due to the C=C stretch typical for cyclohexene. The symmetric and asymmetric O=S=O stretching vibration peaks are located at 1180 and 1359 cm-1, respectively. Other characteristic absorption for acesulfame-K is S-N-C, which appears at 943 cm-1, and out-of-plane
Sodium saccharine spectrum (Figure 2) show characteristic absorption bands at 1647 cm-1 for C=O (Amide I band), at 1150 cm-1 and 1260 cm-1 for symmetric and asymmetric S(=O)2 stretching vibration peaks and S-N-C absorptions, which appears at 975 cm-1. At 1588 cm-1 C-C stretching mode band in benzene ring appears [15], but the Amide II band is
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missing due to amide cis linkage. Characteristic absorptions are also found at 1456 cm-1 for NH bending mode and at 1050 cm-1 for to the C-H in-plane deformation vibration mode (βCH) of the ortodisubstituted benzene [16]. The selection of the FT-IR bands for the aspartame, acesulfame-K, sodium cyclamate and sodium saccharine determination in Formulation I and II considered on the one hand the high specificity of each peak and on the other hand avoided their overlapping in the spectral region of interest. For the percentage
composition determination of the pure sweeteners in Formulation I and II the ratios between peak areas of the specific vibration in ternary formulation and pure compound were calculated, respectively. Therefore, the bands at 836 cm-1 for ACK, at 1736 cm-1 for ASP, at 2854 cm-1 for SCY and at 1050 cm-1 for SSA were selected. In every case, the use of peak area and peak height absorbance was considered. Table 1 and 2 summarize the analytical data of FTIR investigations for determination of the percentage composition of Formulation I and II.
TABLE 1. FT-IR Determination of the Percentage Composition of ACK, ASP and SCY in Formulation I Acesulfame-K (ACK)
Aspartame (ASP)
Sodium Cyclamate (SCY)
Measurement mode
Peak area
Absorbance
Peak area
Absorbance
Peak area
Absorbance
Wavenumber (cm-1) Pure compound Formulation I Percentage (%)
819.6-848.5 2.2948 0.3476 15.15
836 0.1970 0.0210 15.20
1713.4-1768.4 15.1165 1.14220 7.56
1736 0.9237 0.0807 8.73
2825.3-2884.9 6.2594 4.8380 77.29
2854 0.3265 0.2858 87.53
TABLE 2. FT-IR Determination of the Percentage Composition of ACK, ASP and SSA in Formulation II Acesulfame-K (ACK)
Aspartame (ASP)
Sodium Saccharine (SSA)
Measurement mode
Peak area
Absorbance
Peak area
Absorbance
Peak area
Absorbance
Wavenumber (cm-1) Pure compound Formulation II Percentage (%)
828.3-838.9 0.3731 0.0030 0.80
836 0.0935 0.0011 0.12
1722.1 -1768.4 18.8880 1.5683 8.30
1736 0.9177 0.06099 6.64
1041.4 -1054.9 1.0908 0.9930 90.90
1050 0.3878 0.3573 92.14
FIGURE 3. The EI mass spectra of the studied compounds: a) Aspartame, b) Sodium saccharine, c) Acesulfame-K and d) Sodium cyclamate.
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As can be seen, the integration regions for ACK and ASP differ from Formulation I to Formulation II due to the interferences with the SCY and SSA bands. For the comparison of the peak area and peak height measurement modes it can be concluded that the first mode use provides a greater accuracy and reliability.
advantage due to the fact that the exact structure of the compounds can be determined. The cyclamate as sodium salt cannot be seen in the studied mixtures.
ACKNOWLEDGMENTS The financial support of this work by CORE Program is acknowledged.
EI-MS Studies For quantitative analysis of substances mixtures the individual mass spectrum of each compound was obtained. The main ions present in the spectrum were selected for identification and for each compound the base ion was selected for quantification. The mass spectrum of the pure compounds is shown in Figure 3 (a, b, c and d respectively). In the mass spectrum of aspartame the molecular ion is lost but is visible the ion [M-32]+.produced by the elimination of the neutral HOCH3, common process for methyl esters. In the mass spectrum of Acesulfame-K the highest detected ion is to m/z 163 formed by elimination of K and an addition of one H atom. The molecular ion of sodium saccharine was obtained in every situation with intensity above of 90 %. For Sodium cyclamate (Figure 2d) the mass spectrum shows the ions to m/z 217 and 260. By highresolution exact mass measurements it was established that the real structure of the compounds is: C6H11-NHSO2-NH-C6H11 and is not a sodium salt. For quantitative determinations of each compound in the mixtures the ions to m/z 91, 183, 43 and 56 were used as area ratio.
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CONCLUSIONS A rapid and sensitive procedure to estimate the concentration of some non-nutritive sweeteners in ternary formulations using FT-IR (KBr matrix) technique was developed. The FT-IR procedure was validated by EI-MS determination of the samples content. These combined methods require short analysis durations and represent a low cost alternative in the quality control of such sweeteners in ternary formulations for beverages and dietary products. The mass spectrometry methods present a very important
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