Simultaneous determination of benzoic acid and saccharin in soft drinks by using lanthanide-sensitized luminescence M. P. Aguilar-Caballos, A. Gómez-Hens and D. Pérez-Bendito Analytical Chemistry Division, Faculty of Sciences, University of Córdoba, 14004-Córdoba, Spain Received 26th March 1999, Accepted 24th May 1999 A simple and fast approach is used for the first time to develop a time resolved lanthanide-sensitized luminescence method for the simultaneous determination of a preservative and a sweetener, namely benzoic acid (BZ) and saccharin (SC), respectively, in food samples. The method involves the formation of the corresponding ternary chelates with terbium(iii) and trioctylphosphine oxide (TOPO) in the presence of Triton X-100, and the measurement of the initial rate and equilibrium signal of this system, which were obtained in 0.1 and 5 s, respectively. The dynamic ranges of the calibration graphs, obtained by using kinetic and equilibrium measurements, were 0.2–36 mg ml21 and 0.15–30 mg ml21, respectively, for BZ, and 3.3–24 mg ml21 and 4–36 mg ml21 for SC and the detection limits were 0.07 and 0.04 mg ml21, respectively, for BZ, and 1.1 and 1.2 mg ml21, respectively, for sodium SC. The relative standard deviation ranged between 2.3 and 3.0%. Both compounds were determined simultaneously by using a system of two equations which were resolved by using the calibration data obtained individually for each analyte and by multiple linear regression. Mixtures of BZ and SC in ratios between 3:1 and 1:9 were satisfactorily resolved by using both approaches. The method was applied to the direct analysis of several soft drinks. Analytical recoveries ranged between 89.3 and 108.5%. Introduction cence mode so eliminating scatter, Raman and any fluorescence background signal. Benzoic acid (BZ) and saccharin (SC) are two common Benzoate was previously reported as a counter ion in studies additives widely used in numerous foods as a preservative and on lanthanide–crown ether complexes extracted with ethyl an artificial sweetener, respectively. They are considered acetate.12 Later, the possibility of using the benzoate ion for innocuous for humans at the allowed concentration levels, but it energy transfer to the lanthanides in an aqueous medium was was reported1 that benzoates can promote an allergic response studied and applied to the determination of these ions.13 in sensitive people and the potential toxicity of SC, which is Detection limits of 1029 M for terbium and 1028 M for prohibited in some countries considering possible carcinogenic dysprosium and europium were reported. Several methods effects, still continues to be a topic of investigation. The based on terbium-sensitized luminescence have been reported simultaneous determination of these compounds in food for the determination of benzoic acid derivatives such as p- analysis is mainly carried out by using liquid chromatography aminobenzoic, salicylic and p-aminosalicylic acids and di- and UV photometric detection.227 Although these methods flunisal and applied to clinical14,15 and pharmaceutical16 allow other additives together with BZ and SC to be determined, analysis. However, the application of this approach to the some of them involve a solid-phase extraction by using individual and simultaneous determination of BZ and SC and its 224 quaternary ammonium or C18 cartridges prior to the liquid use in routine food analysis have not been described up to date. chromatographic analysis, which causes difficulties in the The proposed method combines the selectivity of lanthanide- application of these methods to routine food analysis. Thus, the sensitized luminescence with kinetic and equilibrium measure- main aim of this paper has been the development of a simple, ments of both BZ and SC systems. The high initial rate of these automatic and fast method for the simultaneous determination reactions requires the use of stopped-flow mixing technique, of BZ and SC, which does not require any previous separation which also enables ready automation of the method since step and can be easily applied to the analysis of soft drink reactant manipulation is minimal and measurements are samples. performed shortly after mixing. In spite of the high sensitivity of photoluminescence techniques, they have been rarely applied to the individual determination of BZ8 and SC9 and their simultaneous determi- nation has not been described up to date. Thus, the lack of Experimental luminescent methods for the resolution of mixtures of these compounds led us to study their reactions with lanthanide ions. Instrumentation As known, many lanthanide chelates feature a special lumines- cent behaviour as a result of the intramolecular energy transfer An SLM-Aminco (Urbana, IL, USA) AB2 luminescence process from the ligand to the central ion, which avoids or spectrometer, equipped with a 150 W continuous xenon lamp minimizes selectivity limitations of luminescence methods.10,11 and a 7 W pulsed xenon lamp, was used. The instrument was They show large Stokes shifts and narrow emission bands, furnished with a stopped-flow module17 supplied by Quimi-Sur which allow the spectral discrimination of the analytical signal. Instrumentation (Seville, Spain), which was fitted with an Also, their relatively long lifetimes allow the temporal discrim- observation cell of 1 cm path length. The excitation and ination by making analytical measurements in the phosphores- emission slits were adjusted to provide an 8 nm band-pass. The Analyst, 1999, 124, 1079–1084 1079 temperature of the solutions in the stopped-flow module and cell compartment was kept constant at 25 °C by circulating water from a thermostated tank. Reagents All chemicals used were of analytical reagent grade. Stock solutions of BZ (100 mg ml21) (Sigma, Poole, Dorset, UK) and SC (400 mg ml21) (Sigma) were prepared in ethanol–distilled water (1 + 99). A 2 3 1022 M terbium(iii) solution was made by dissolving an appropriate amount of terbium(iii) nitrate penta- hydrate (Aldrich, Gillingham, Dorset, UK) in distilled water. A 6 3 1024 M solution of TOPO (Sigma) was prepared in 50% ethanol. An 1% Triton X-100 (Fluka, Buchs, Switzerland) aqueous solution and an imidazol buffer solution (0.2 M, pH 6.0) were also used. Fig. 1 Kinetic curves obtained for SC (1), BZ (2) and a mixture of both (3) in the presence of terbium(iii) (2 3 1023 M), Triton X-100 (8 3 1022 M) 25 21 21 and TOPO (5.6 3 10 M). [SC] = 10 mg ml , [BZ] = 1 mg ml . lex 284 nm, lem 545 nm (IL = luminescence intensity). Procedures measuring the initial rate (v) and equilibrium signal (IL) of the Individual determination of BZ and SC. A solution kinetic curve and applying the following equations: containing terbium(iii) (2 3 1023 M), Triton X-100 (8 3 22 25 22 10 %), TOPO (5.6 3 10 M), imidazol buffer (2.4 3 10 v = a0 + a1[BZ] + a2[SC] M) and ethanol (4%) was used to fill one of the two 2 ml drive IL = aA0 + aA1[BZ] + aA2[SC] syringes of the stopped-flow module. The other syringe was filled with a solution containing standard or sample BZ or SC, where [BZ] and [SC] are the concentrations of the analytes imidazol buffer (2.4 3 1022 M) and ethanol (4%). The final expressed in mg ml21. Two methods were used to resolve this 21 concentration ranges of BZ and SC were 0.2–30 mg ml and equation system. In the first method (conventional method), a0 21 4–36 mg ml , respectively, in the equilibrium method, and and aA0 are the sum of the intercepts of the corresponding 0.3–36 mg ml21 and 4–34 mg ml21, respectively, in the kinetic calibration graphs obtained individually for each compound by method. In each run, 0.15 ml of each solution was mixed in the using the initial rate or the equilibrium signal as the analytical mixing chamber. The variation of the luminescence intensity parameter, and a1, a2, aA1 and aA2 are the corresponding slope of with time throughout the reaction was monitored at lex 284 nm each calibration graph; the equations were resolved by using a and lem 545 nm by using the phosphorescence mode, with delay simple BASIC program. In the second method, the coefficients and gate times of 0.1 and 5 ms, respectively. Kinetic data were of these equations were estimated by multiple linear regression processed by linear regression using the microcomputer, (MLR) from 20 mixtures of both analytes in the above furnished with a program for application of the initial-rate established ranges, and the system was resolved by using a method. The dynamic and equilibrium signals were measured in laboratory-developed FORTRAN 77 program.18 ca. 0.1 s and 5 s, respectively. All measurements were made at 25 °C and each standard or sample was assayed in triplicate. Determination of BZ and SC in soft drink samples. Each Although the contribution of the blank signal was very low, it sample (2.5 ml) was degassed, neutralised with 2 M sodium was subtracted from the kinetic and equilibrium measure- hydroxide and diluted to 10 ml with distilled water. A volume ments. (1 ml) of this solution was treated as described above. Simultaneous determination of BZ and SC. The procedure used was the same as that described above, but the syringe Results and discussion containing the analyte was filled with appropriate amounts of both compounds to obtain a final concentration level of each In order to choose the adequate systems for the determination of analyte within the corresponding calibration graph. The concen- BZ and SC, based on lanthanide-sensitized luminescence, a tration of each analyte in the mixture was calculated by series of previous assays were carried out, taking into account Fig.