ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
DETERMINATION OF BENZOATE, SORBATE, CITRATE AND ORTHOPHOSPHATE IONS IN BEVERAGE SAMPLES USING TWO-DIMENSIONAL ISOTACHOPHORETIC METHOD © 2012 г. Aneta Jastrzgbska, Marzanna Kurzawa, Pawe l Hrynczyszyn, Edward Szl yk
Faculty of Chemistry, Nicolaus Copernicus University 7 Gagarin Str., 87-100 Torun, Poland Received 03.06.2010; in final form 07.04.2011
A two-dimensional capillary isotachophoretic method (cITP-cITP) using electrolyte system consisting of leading electrolytes (LE1): [10 mM HCl + P-alanine (pH 3.9) + 0.1% hydroxyethylcellulose (HEC)] and (LE2): [10 mM HCl + aminocaproic acid (pH 5.00) + 0.1% HEC], and 5mM caproic acid as terminating electrolyte (TE) was studied. Two methods of detection, conductometric and UV-Vis, were applied to the determination of selected food preservatives and additives. Practical applicability was demonstrated by simultaneous determination of benzoates, sorbates, citrates and orthophosphates in 12 samples of beverages. The proposed method revealed linearity with R2 between 0.9992 and 0.9999 for the concentration ranges: 10—100 mg/L (orthophosphate and citrate ions), 20—100 mg/L (sorbates) and 40—120 mg/L for benzoates. The detection limits for all studied ions were from 0.85 to 3.1 mg/L whereas the quantification ones were from 2.8 to 10 mg/L. The variation coefficients for five-fold analysis of all ions ranged between 0.4 and 9.1%. Obtained recoveries (from 97 to 104%) confirmed satisfactory accuracy of the proposed cITP-cITP method for the determination of tested food additives.
Keywords: cITP-cITP method, HPLC, samples of beverages, benzoates, sorbates, citrates, orthophosphates.
Chemical preservatives have played an increasingly important role in food industry, since they are used to prevent the growth of bacteria, yeasts and molds in food [1]. The most popular preservatives are sorbic or benzoic acids and their salts. These compounds exhibit inhibitory activity against wide varieties of fungi, yeasts, molds and bacteria, including most food borne pathogens. A broader spectrum of microbicidal activity is often achieved by using a combination of preservatives [2]. However, excessive addition of these preservatives may be harmful to consumers, due to the possible allergic contact dermatitis, convulsion, hives, etc. [3]. The use of sorbic and benzoic acids, as well as their salts is approved by European and Polish legislation and its presence must be declared on the label. These antimicrobial agents are often used in conjunction with natural preservative like citric acid. The organic acids are natural components of many fruits and vegetables and they play an important role in maintaining food quality due to inhibition of bacteria growth. In addition to the pH-lowering effects of citric acid, a secondary inhibitory effect is attributed to the chelation of essential minerals. Citric acid is generally recognized as safe with no limitations when used in accordance with good manufacturing practice.
A variety of analytical methods are available for the determination of benzoates, sorbates and citrates. Most of the reported methods are based on chromatographic methods such as high-performance liquid
chromatography (HPLC) [2, 4—7], gas chromatography (GC) [8—12], micellar electrokinetic chromatography [13—15] and ion chromatography (IC) [16—18]. Nowadays, electrophoretic methods have become an attractive analytical technique for preservatives due to its high separation efficiency, low sample consumption and short analysis time [3, 19—23].
Recently, much attention has been paid to the search for effective and reliable analytical methods of preservatives determination in food with reduced sample preparation techniques. Capillary isotachophore-sis (cITP) is an effective tool for analysis of ionic compounds in various matrices. The concentration adaptations of the analytes are characteristic for cITP method. Furthermore, the electroosmotic flow is completely suppressed by the use of a hydrodynami-cally closed system, capillaries of inert polymers and additives in the electrolyte system [24]. The on-line preconcentration of weak electrolytes by capillary electrophoresis (CE) in comparison with isota-chophoretic techniques for sorbic and benzoic acids was described by Horakova et al. [25]. Isotachophoretic determination of 13 organic and inorganic acids in wines on a poly(methyl methacrylate) chip was discussed by Masar et al. [26].
Concentration of analytes, accompanied by a well-defined sample clean-up, is probably the main benefit of two-dimensional capillary isotachophoresis
(cITP-cITP). In this system preseparation column enables a considerable reduction of the matrix effect onto the analytical column, while the concentrated analytes are transferred for the final separation [27]. The application of cITP-cITP technique provides an assay of complex samples during one analysis in short time. The present study is intended to develop a simple and reliable cITP-cITP method with a conductomet-ric and UV-Vis detectors for simultaneous determination of selected food additives. The conductometric detection is a key detection technique in cITP and is currently employed in conventional CE separation systems. In this paper we applied cITP-cITP method with two detection systems (conductometric and UV-Vis) to simultaneous determination of benzoate, sorbate, citrate and orthophosphate ions in beverages after simple dilution in an appropriate ratio. It should be noted that the proposed electrolyte system permitted on determination of organic and inorganic compounds during one measurement. For this reason, elaboration of inexpensive method for routine analysis of tested food additives in quality and assurance control laboratories was the main goal of our work. The results obtained for benzoate and sorbates ions were compared with those obtained by HPLC method. In addition, to confirm the reliability of the method standard solution of each ion was added to the food sample, and recovery was tested.
EXPERIMENTAL
Reagents and apparatus. Analytical grade P-alanine (BALA), hydroxyethylcellulose (HEC), aminocaproic acid, caproic acid and acetonitrile (for HPLC) were purchased from Sigma Aldrich, whereas C6H7KO2, NaC6H5CO2, C6H8O7 KH2PO4, CH3COOH, CH3COONH4, CH3COONa, and HCl were from Al-chem (Poland). Redistilled water was used in all solutions preparation (specific conductivity <10 ^S).
Isotachophoretic separations were performed using a Villa Labeco EA 100/101 isotachophoretic analyzer equipped with a conductometric detector and UV-Vis detector (Sapphire UV-VIS for HPLC). The PTFE pre-separation capillary (90 mm x 0.8 mm I.D.) was connected with PTFE analytical capillary (160 mm x x 0.3 mm I.D.). Samples of 30 ^l fixed volume were injected via a sample valve by internal sample loop. The isotachopherograms were evaluated with the software supplied with analyser (KasComp Ltd., Slovakia). HPLC analyses were performed with a Shimadzu chromatograph C-R6A Chromatopac with UV-VIS detector and a column C8 (15cm x 0.46 cm), Supelco Discovery, Germany.
Sample preparation. Five juice samples (marked as 1—5) and seven soft drinks (marked as 6—12) were purchased from local markets. In all samples benzoates, sorbates and citrates in composition were declared by the producers. Some of the samples contained phos-
phoric acid. The tested beverages were diluted in an appropriate ratio (1/2; 1/4 or 1/20) with redistilled water (for isotachophoretic method) or mobile phase (for chromatographic method).
Determination of benzoates, sorbates, citrates and orthophosphates by cITP-cITP. Analysis of benzoate, sorbate, citrate and orthophosphate ions were performed with LE1: 10 mM HCl + p-alanine (pH = 3.9) + 0.1% HEC and LE2: 10 mM HCl + aminocaproic acid (pH 5.00) + 0.1% HEC. 5 mM caproic acid was applied as TE. A driving current of the preseparation capillary was 200 ^A. In the case of analytical capillary, driving current was 50 ^A which during detection decreased to 30 ^A. The UV-Vis detection was at 254 nm for benzoate and sorbate ions.
Determination of benzoates and sorbates by HPLC.
The measurement at X 252 nm (sodium benzoate) and X 256 nm (potassium sorbate) were carried out using the mixture of 0.005 M ammonium acetate + 15% ac-etonitrile + acetic acid to pH 4.0 as a mobile phase. The flow-rate was 1.1 mL/min for sodium benzoate and 1.3 mL/min for potassium sorbate.
Calibration curves for sodium benzoate and potassium sorbate were constructed using six calibration solutions of tested ions. Results were calculated as an average of five replicates. The calibration curve for sodium benzoate, expressed as y = (b ± Sb)x + (a ± Sa), where Sb, Sa are standard deviations of the slope and intercept, was found resulting in the regression equation of y = (8736 ± 64)x + (65 076 ± 4235) with the determination coefficient R2 = 0.9986. The detection (DL) and quantification limits (QL) were calculated according to Miller and Miller [28], the obtained results being as follows: DL = 3.25 mg/L and QL = = 10.8 mg/L. The calibration equation for potassium sorbate was y = (158438 ± 2829)x + (181157 ± 14368) with the correlation coefficient 0.9960, whereas DL = = 6.08 mg/L and QL = 20.3 mg/L.
RESULTS AND DISCUSSION
The benzoate and sorbate ions were identified by migration time, whereas the orthophosphate and citrate anions were identified using the relative step height (RSH) parameter. Precision was evaluated as the within-day and between-days coefficient of variation (CV) [28]. Within-day analyses were determined by injection of the standard solutions five times per day. Intralaboratory reproducibility was determined by analysis of the standard solutions during 5 consecutive days. The obtained results are presented in Table 1.
Data in Table 1 exhibited that orthophosphate and citrate ions were separated in preseparation column and could be quantified by conductometric detection. Furthermor
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