Analytica Chimica Acta 535 (2005) 5763

EDTA determination in pharmaceutical formulations and canned foods based on ion chromatography with suppressed conductimetric detection
A.A. Krokidis, N.C. Megoulas, M.A. Koupparis
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece Received 21 October 2004; received in revised form 6 December 2004; accepted 6 December 2004 Available online 19 January 2005

Abstract A novel direct method for the determination of EDTA was developed and validated based on ion chromatography with suppressed conductimetric detection and anion exchange column (Dionex AS-14, 4 mm 250 mm). Depending on coexisting substances, suitable eluents are 10 mM carbonate buffer/pH 11.0 or 10.5 (tR,EDTA = 5.5 and 9.4 min, respectively), and 120 mM borate buffer/pH 8.5 (tR,EDTA = 16.2 min). For 10 mM carbonate buffer/pH 11.0 and isocratic ow rate of 1.0 ml min1 , a linear calibration curve was obtained from 2.7 to 100 g ml1 (r > 0.998), with LOD 0.87 g ml1 and %RSD 1.5 (5 g ml1 , n = 9). Good resolution was achieved from commonly coexisting anions (chloride, metabisulphite, ascorbate and citrate), and other aminopolycarboxylic acids (EGTA, NTA and DTPA). The potential interference of pharmaceutical substances (caffeine, phenytoin, nembutal, tolbutamide, dicumarol, acetylsulphisoxazole and paracetamol) and metal cations (Ca2+ , Cu2+ and Fe3+ ) was also examined. The ion chromatographic method was applied for the assay of EDTA in contact lens care solutions, synthetic injection drug solutions, canned mushrooms and mayonnaise, with simple treatment and good recovery (range 74108%). 2004 Elsevier B.V. All rights reserved.
Keywords: EDTA; Ion chromatography; Suppressed conductimetric detection; Pharmaceuticals; Foods

1. Introduction Ethylenediaminetetraacetic acid (EDTA) is a powerful chelating agent, forming stable complexes with most metal ions. Due to its ability to sequester metal ions, EDTA is widely used in medicine, chemical industry, food technology, agriculture and pharmaceutical technology. In medicine, it is used for the treatment of lead poisoning [1] and in agriculture to enhance the delivery of metal micronutrients in plants [2]. Its industrial applications include pulp, paper, metal and textile production [3]. EDTA, in its disodium salt or calcium disodium salt form, is frequently added in pharmaceutical formulations and foods, because of its stability, compatibility and low toxicity. In pharmaceutical formulations it enhances the action of preservatives and antibacterials and stabilizes the

Corresponding author. Tel.: +30 210 7274559; fax: +30 210 7274750. E-mail address: koupparis@chem.uoa.gr (M.A. Koupparis).

action of antioxidants [4,5]. In foods (e.g. canned or pickled vegetables, canned mushrooms, mayonnaise and salad dressings) EDTA is added to prevent deteriorative changes, and to preserve color, odor and avor. Its action is based on the chelation of metal ions, which catalyze oxidation reactions, and the inactivation of enzymes that cause enzymatic browning [6,7]. At the same time, usage of EDTA or other sequestrants is strictly regulated by U.S. Food and Drug Administration or relevant agencies. In the eld of analytical chemistry, besides its use in complexometric titrations, EDTA has been reported to be a very useful ligand for the complexation of metals, which enables their chromatographic separation [8]. Various analytical methods have been proposed for the determination of EDTA in a wide variety of sample matrices and reviewed [9]. They include titrimetry [10], spectrophotometry [11], electrochemistry (e.g. polarography [12], differential pulse polarography [13], catalytic potentiometric titrimetry [14], one-drop square-wave polarography [15],

0003-2670/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.12.011

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differential pulse anodic stripping voltammetry [16], amperometry [17]), capillary electrophoresis [18] and chromatography. Amongst them, gas chromatography and HPLC (reverse-phase ion-pair or ion exchange retention mechanism) appear to be the prevailing techniques, despite the fact that EDTA lacks volatility and exhibits low UV/Vis absorptivity. The gas chromatographic methods always include a time-consuming derivatization step, in which EDTA is converted into methyl, ethyl, propyl or butyl esters to obtain volatility [1921]. Similarly, HPLC methods are mainly based on a pre-column derivatization step in which complexation of EDTA with Cu2+ or Fe3+ is conducted [5,6,2224], while post-column derivatization for the formation of uorescent ternary complexes has also been reported [25]. This paper describes a systematic study of the ion chromatographic behavior of EDTA and the effect of various substances, such as inorganic and organic ions (Fe3+ , Cu2+ , Ca2+ , Cl , S2 O5 2 , ascorbate, citrate), similar aminopolycarboxylic acids with complexing properties (EGTA, NTA, DTPA) and pharmaceutical substances. Based on this study, a novel ion chromatographic method with suppressed conductimetric detection for the direct (without derivatization step) determination of EDTA was developed and validated. The method was subsequently used for the determination of EDTA in pharmaceutical formulations (contact lens care solutions and synthetic injection solutions) and canned foods (mushrooms and mayonnaise).

thetic injection drug solutions (containing 500 g ml1 of drug [caffeine, phenytoin, nembutal, tolbutamide, dicumarol, acetylsulphisoxazole, paracetamol] and 100 g ml1 EDTA in NaCl 0.9%) were also prepared to be analyzed. 2.2.1. EDTA standard solutions An EDTA stock solution (equivalent to free acid 500 g ml1 ) was prepared by dissolving the appropriate amount of EDTA disodium salt (C10 H14 N2 Na2 O8 2H2 O, Riedel-de-Ha en, extra pure) in water and stored in the refrigerator (at 4 C) in a plastic bottle. Working standard solutions (in the range of 2.7100 g ml1 ) were daily prepared by appropriate dilution with mobile phase. 2.2.2. Mobile phase The selected mobile phases (10 mM aqueous carbonate buffer with pH 10.5 or 11.0) were prepared by dissolving sodium hydrogen carbonate in water and adjusting the pH with a concentrated sodium hydroxide solution. They were stored in the refrigerator in a plastic bottle and renewed every 2 weeks. Other examined mobile phases were: (i) 2032 mM borate aqueous solutions (Na2 B4 O7 10H2 O), without pH adjustment, (ii) 28150 mM borate buffers of pH 8.59.5 (H3 BO3 + NaOH), and (iii) 720 mM carbonate buffers of pH 9.511.0. 2.3. Procedures

2. Experimental 2.1. Instrumentation Ion chromatographic separations were carried out on the Dionex DX-100 ion chromatographic system, consisting of: a DX-100 high pressure one piston pump, a sample injector equipped with a 25 l loop, an Ion pac AG14 guard (4 mm 50 mm) and an AS-14 analytical column (4 mm 250 mm) (macroporous particles with 9.0 m total capacity of 78 eq), diameter, porous size 100 A, an ASRS-I micro-membrane suppressor operating in the auto suppression recycle mode (selectable current intensity 50500 mA), and a conductimetric detector (dead volume 1.25 l) equipped with a thermistor for the compensation of temperature variations. The chromatographic peaks were electronically integrated and recorded with a Hewlett Packard model 3395 integrator. 2.2. Reagents All chemicals were of analytical reagent grade and used without further purication. HPLC-grade water was obtained with a Milli-Q water purication system (Millipore). The analyzed canned foods and pharmaceutical formulations were obtained from local commercial sources. Syn-

The chromatographic elution was performed at room controlled temperature (2224 C) in isocratic mode at 1.0 ml min1 ow rate. The selected current intensity for the suppressor operation was 300 mA. The eluent solutions were ltered through a 0.45 m membrane lter (HVLP, Millipore) before usage. The ow path was rinsed for about 15 min, until baseline noise became less then 0.1 S. Other instrumental parameters were: air pressure 5 psi and conductivity range 30 S. Quantitation was based on peak areas. 2.3.1. Sample preparation Liquid samples (contact lens care solutions, injection drug solutions and surrounding liquid of canned mushrooms) were diluted with the eluent, ltered, and injected into the chromatographic system without further treatment. Mayonnaise samples were mixed with water, mildly heated, ltered and extracted with benzene, for the removal of the fatty substances. In the case of coexistence of organic drugs that are adsorbed on the polymeric column (e.g. paracetamol), removal of the drug molecules, using liquidliquid extraction with ethyl acetate is required before injection to the chromatograph. In the case of coexistence of surfactants (e.g. contact lens care solutions), a periodical washing of the analytical column with acetonitrile is required.

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3. Results and discussion 3.1. Selection of mobile phase EDTA (H4 Y) is a weak tetraprotic carboxylic acid. Five EDTA species (H4 Y, H3 Y , H2 Y2 , HY3 and Y4 , dissociation constants: K1 = 1.02 102 , K2 = 2.14 103 , K3 = 6.92 107 and K4 = 5.50 1011 ) coexist in aqueous solutions and their concentration distribution depends on the pH of their environment (mobile phase). The electric charge of EDTA species strongly inuences its selectivity coefcient (retention) but not the conductimetric detector response factor, since electrochemical suppression was occurred before detection. Preliminary experiments were carried out utilizing borate (Na2 B4 O7 10H2 O) eluents in the concentration range of 10.032.0 mM, without pH adjustment. In all cases, two chromatographic peaks were recorded for EDTA. The existence of two peaks in the EDTA chromatograms may be attributed to the formation of EDTA complex with Na+ . Despite the low formation constant of NaY3 complex (log kf = 1.7), the relatively high concentration of Na+ in mobile phase favors the complex formation. Therefore, the rst chromatographic peak may be attributed to the NaY3 complex, while the second one to the free EDTA species. Increase of borate concentration in mobile phase resulted in decrease of retention time of both peaks. The EDTA ion chromatographic behavior was further investigated using borate (H3 BO3 + NaOH) mobile phases (28112 mM) with adjusted pH within the range of 8.510.0 (dominant EDTA species HY3 and Y4 ). It was found that a decrease of pH of mobile phase resulted in a decrease of the area of the rst peak (NaY3 ), which was accompanied by an increase of the area of the second peak. A decrease of pH, along with the decrease of sodium concentration, shifted the protonation equilibrium among the various EDTA species in favor of HY3 , which suppressed the formation of NaY3 complex. Therefore, the area of the rst peak decreased, while an increase was observed for the second peak. For pH equal to 8.5, the rst peak was disappeared, obtaining only one chromatographic peak (Fig. 1). In the contrary, for pH above 10.5, the Y4 species dominated, the concentration of sodium in mobile phase (the counter cation of hydroxides) increased, and so the formation of NaY3 complex was favored inducing the elimination of the second and an increase of the rst chromatographic peak. This effect was observed for carbonate mobile phases (720 mM) with varying pH within the range of 9.511.0. For 10 mM carbonate mobile phase with pH = 10.5, the ratio of the area of the rst peak to the area of the second peak was found to be 25.3. An overall evaluation of the results from this study leads to the conclusion that the ion chromatographic method for the determination of EDTA should be based on mobile phase with pH below 8.5 (borate buffer, dominant species HY3 ) or above 10.5 (carbonate buffer, dominant species NaY3 ).

Fig. 1. Effect of pH of borate mobile phase on the EDTA chromatographic peaks: (A) pH = 10 (the rst peak (tR = 10.6 min) corresponds to NaY3 complex, and the second one (tR = 16.9 min) to free EDTA species), (B) pH = 9.5 (the rst peak (tR = 11.6 min) corresponds to NaY3 complex, and the second one (tR = 18.2 min) to free EDTA species) and (C) pH = 8.5 (the rst peak was disappeared and only one peak corresponding to HY3 species was recorded, tR = 17.4 min).

Calibration curves for EDTA in the range of 5100 g ml1 were constructed in order to evaluate the analytical characteristics of various mobile phases. Table 1 summarizes the chromatographic and analytical characteristics of each tested mobile phase. As it is shown, an increase of carbonate or borate concentration was accompanied by a considerable decrease in the retention time of EDTA. The symmetry of the EDTA peak (asymmetry factor 1.11.3) was suitable for all eluents tested. The resolution from chloride ions (common excipient) in all cases was excellent, while the resolution from metabisulphite (S2 O5 2 ) was better for carbonate mobile phase (Rs = 1.7) (Fig. 2). Comparing all the chromatographic and analytical characteristics [analysis time, peak symmetry, resolution between adjacent peaks, sensitivity (slope of calibration curve), detectability (detection limit) and precision], it was concluded that a 10 mM carbonate buffer/pH 11.0 is the optimum eluent (tR = 5.5, LOD = 0.87 g ml1 ).

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Table 1 Chromatographic and analytical characteristics of EDTA determination (5100 g ml1 ) for the selection of mobile phase Mobile phase tR (min) Retention factor, k Asymmetry factor Resolution, REDTA/S2 O5 2 Slope of calibration curve (105 V s ml mg1 ) 1.64 (0.03) 1.90 (0.04) 2.18 (0.07) 2.85 (0.12) 2.55 (0.02) 2.63 (0.16) Correlation coefcient (n = 5) 0.998 0.998 Detection limit (g ml1 ) 1.2 0.87 %RSD (n = 3 3)a 2.3 g ml1 3.0 1.5

Carbonate buffer 8 mM, pH 10.5 9.4 10 mM, pH 10.5 6.3 10 mM, pH 11.0 5.5 Borate buffer H3 BO3 + NaOH, pH 8.5 112 mM 17.0 120 mM 16.2 140 mM 12.5 150 mM 11.8
a

9.4 6.0 5.1 17.9 17.0 12.9 12.1

1.3 1.2 1.1

1.7 1.4

1.3 1.3 1.1 1.1

1.0 1.0 1.0 0.9

0.997 0.993 0.999 0.996

4.2 0.53 0.22 0.83

1.5 1.5 0.7 2.6

Three working days (within a week), three injections per day.

3.2. Separation from potential interfering substances Interferences from substances usually coexisting with EDTA in foods and formulations were studied, using the carbonate eluent. It was examined, whether ascorbate and citrate, common preservatives for canned foods, affect the chromatographic behavior of EDTA. The applied mobile phase was the 10 mM carbonate buffer (pH = 10.5). No interference was observed for both molecules, since the retention time of citrate was 52.1 min, while EDTA resolution from ascorbate was Rs = 1.2.

Potential interferences from chelating aminocarboxylic acids [EGTA (ethyleneglycol-bis-(2-aminoethylether)tetraacetic acid), NTA (nitrilotriacetic acid) and DTPA (diethylenetriaminepentaacetic acid)] were also examined. DPTA appeared retention time logger than 30 min (it bears an extra carboxylate group in comparison to EDTA) and it was not further investigated. NTA and EGTA were eluted at 6.9 and 7.4 min, respectively, resulting in inadequate resolution from EDTA (tR = 6.3 min). Better separation can be achieved by reducing the carbonate concentration in mobile phase. Utilizing an 8.0 mM carbonate eluent (pH = 10.5), the retention time of EDTA was 9.4 min, while the retention time of NTA and EGTA were 12.7 and 15.1 min, respectively. Resolution between EDTA and NTA was 1.2 (Fig. 3). Calibration graph was constructed with

Fig. 2. Resolution of EDTA (5 g ml1 ) from chloride (100 g ml1 ) and metabisulphite (20 g ml1 ) with 10.0 mM carbonate eluent (pH = 10.5) [tR = 3.7 min corresponds to chloride, tR = 6.4 min corresponds to EDTA and tR = 8.0 min corresponds to metabisulphite].

Fig. 3. Resolution of EDTA from NTA, EGTA and DPA, all at a concentration level of 100 g ml1 , with 8.0 mM carbonate eluent (pH = 10.5) [tR = 9.4 min corresponds to EDTA, tR = 12.7 min corresponds to NTA and tR = 15.1 min corresponds to EGTA, no peak was recorded for DPA].

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EDTA standard solutions containing 100 g ml1 of NTA and EGTA. No signicant change was found to be caused by NTA or EGTA in EDTA peak area and in the slope of the calibration curve (t-test). Since the scope of our study was mainly the application of the IC method for the determination of EDTA in pharmaceutical formulations, seven pharmaceutical substances of different molecular type (caffeine, phenytoin, nembutal, tolbutamide, dicumarol, acetylsulphisoxazole and paracetamol) were tested for their effect on the EDTA peak. It was found that conductimetric detector appear no response for these substances, since none of them bear signicant electric charge. However, poor symmetry of EDTA peak was observed in case of coexistence of paracetamol (N-4hydroxyphenyl acetamide). Probably paracetamol adsorbed to the polymeric stationary phase reducing the column capacity. It was found that paracetamol can be removed from the sample by liquidliquid extraction with ethyl acetate. The quantity of the extracted paracetramol was monitored using the UV absorption spectrum (paracetamol absorption peak at 280 nm). When 500 g ml1 paracetamol were added to a 50 g ml1 EDTA standard, ve sequential extractions with 10 ml ethyl acetate were required for the complete removal of the interfering substance. Efforts to remove paracetamol utilizing Sep-pac (Waters) C8 and C18 cartridges and the hydrophobic resin Amberlite XAD-2 were unsuccessful. Since EDTA forms strong complexes with metal cations, the chromatographic determination is expected to suffer from strong interference in the presence of divalent or trivalent cations. Investigation was conducted with borate eluent, in order to avoid precipitation of the cations as carbonate salts. Increasing amounts of Ca2+ (Kf = 10.7), Cu2+ (Kf = 18.8) and Fe3+ (Kf = 25.1) were added to a 50 g ml1 EDTA standard solution. Application of a t-test (at 95% condence level) showed that the presence of Ca2+ , Cu2+ and Fe3+ in the sample, at a ratio (EDTA:metal) equal or less than 1:50, 1:10 and 1:1, respectively, has no signicant effect on the EDTA peak area. The results from this study leads to the conclusion that depending on coexisting substances, suitable eluents for the determination of EDTA appears to be 10 mM carbonate buffer/pH 10.5 (tR,EDTA = 6.3 min) or pH 11.0 (tR,EDTA = 5.5 min), 8 mM carbonate buffer/pH 10.5 (tR,EDTA = 9.4 min) and 120 mM borate buffer/pH 8.5 (tR,EDTA = 6.3 min). 3.3. Application to pharmaceutical formulations and canned foods The proposed ion chromatographic method was further applied for the determination of EDTA in pharmaceutical formulations (contact lens care solutions, synthetic injection drug solutions) and canned foods (mayonnaise and preservation liquid of mushrooms). For liquid samples the only required sample treatment was the appropriate dilution with the mobile phase, while for mayonnaise a procedure based

on liquidliquid extraction for the elimination of lipophilic substances was conducted (according to Section 2.3.1). In the case of coexistence of organic molecules that are adsorbed on the analytical column (e.g. paracetamol), extraction with ethyl acetate is required before sample injection. Aqueous 10 mM carbonate buffer (pH = 11.0) was the selected mobile phase. A typical chromatogram of the contact lens care solution is shown in Fig. 4. A variety of viscosity agents, preservatives, surfactants and active drugs were contained in the selected samples. The compositions reported on the product labels are included in Table 2. Retention times of most excipients were examined in order to assure that they do not interfere (peak overlapping) with EDTA. However, since some of them (e.g. surfactants) were excessively retained by the analytical column (due to strong interactions with the polymeric ion exchange stationary phase), their elution was not feasible by the selected mobile phase and therefore, a periodical washing with acetonitrile was performed.

Fig. 4. Typical chromatogram of contact lens care solution with 10.0 mM carbonate eluent (pH = 11.0) [tR = 3.3 min corresponds to chloride and tR = 5.2 min corresponds to EDTA].

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Table 2 Recovery of EDTA from pharmaceutical formulations and canned foods Sample type/declared excipients Oxysept 2 Allergan, contact lens care solution [isotonic solution containing catalytic neutralizer 0.2% (w/v), thiomersal (disinfectant) 0.001%, w/v] Renu Bausch&Lomb, contact lens care solution [isotonic solution containing boric acid, sodium borate, DYMED (polyamino-propylbiguanidine) 0.0005% (w/v) and poloxamine 1%, w/v] Synthetic drug injection solutions of 500 g ml1 (caffeine, phenytoin, nembutal, tolbutamide, dicumarol, acetylsulphisoxazole or paracetamol) [containing NaCl 0.9% and EDTA 100 g ml1 ] Canned mushrooms [sodium chloride, citric acid, ascorbic acid] EDTA found (%, w/v) (n = 5) 0.560 0.007 Fortication levela (g ml1 ) 10 30 50 70 20 30 50 70 % Recovery 105 95 99 97 96 102 98 101 % Mean recovery (S.D.) 99 (4.3)

0.087 0.002

99 (2.8)

97108

0.095 0.002

10 30 50 70 10 40 70 100

74 77 85 80 94 93 90 96

79 (4.7)

Mayonnaise [vegetable oils 76%, eggs 8.5%, vinegar, sodium chloride, sugar, lemon juice, spices]

n.p.b

93 (2.5)

On diluted sample working solution containing about 30 g ml1 EDTA; in the case of mayonnaise the solid sample (250 mg) was dispersed in EDTA solution. b Not present.
a

The accuracy of the new method was evaluated performing recovery experiments by spiking sample working solutions. Recovery ranged from 95 to 105% for pharmaceutical formulations and from 74 to 96% for canned foods (Table 2), which revealed sufcient accuracy. Further study of the matrix effect on the determination was carried out by dilution experiments (determination of EDTA content using a varying dilution factor D (Vinitial /Vnal ) at four different levels). The correlation curves of the concentration found (in the diluted solution) versus D were linear (r > 0.998) with a slope equal to the content of the EDTA and a statistically (proven by t-test) zero intercept. Similarly, the correlation curves of EDTA content found (which corresponds to the initial undiluted sample) versus D were very linear with statistically (proven by t-test) zero slopes. These results conrmed the absence of any constant or proportional determinate error due to matrix (excipients) effect.

this is the rst work dealing with an IC method with suppressed conductimetric detection for the assay of EDTA and can also be applied to the assay of other chelating agents with similar (polyaminocarboxylate) structure.

Acknowledgements We gratefully acknowledge support from the Ministry of Industry, Energy and Technology, General Secretariat of Research and Technology of Greece and the Ministry of Education (EPEAEK II Program Pythagoras).

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