Synthesis, Characterization, Biological Activity and

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synthesis complex, the linear response was within 1x10-6-3x10-5 M with a detection limit ...... beta-lactam antibiotics cefazolin, cefadroxil, cephalexin, ampicil-.

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Current Analytical Chemistry, 2010, 6, 316-328

Synthesis, Characterization, Biological Activity and Voltammetric Behavior and Determination of Cefaclor Metal Complexes Derya Tarinc1, Burcu Dogan-Topal2, Mustafa Dolaz1, Aysegul Golcu*,1 and Sibel A. Ozkan2 1

Kahramanmaras Sutcu Imam University, Faculty of Science and Arts, Department of Chemistry, Campus of Avsar, 46100 Kahramanmaras, Turkey; 2Ankara University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Tandogan-Ankara, Turkey Abstract: Cefaclor (CEF), a second generation cephalosporin antibiotic, possesses various donor sites for interaction with transition metal (II) ions such as Cu(II), Co(II) and Ni(II) to form complexes of the type [M(CEF)(H2O)Cl], with a molar ratio of metal:ligand (M:L) of 1:1. These complexes were prepared and characterized by physicochemical and spectroscopic methods. Their UV-Vis, IR and mass spectra suggest that CEF potentially acts as a bi-dentate ligand. The electrochemical behavior of these synthesized and in solution complexes is studied over glassy carbon electrode in various buffer solutions using cyclic, linear sweep, differential pulse (DP) and square wave (SW) voltammetric techniques. CEF enrichment is observed over Cu(II) complex. The peak current and peak potential of the complex depend on pH, initial potential, and scan rate. DP and SW voltammetric techniques were used for the determination of CEF-Cu(II) complex. For solid synthesis complex, the linear response was within 1x10-6-3x10-5 M with a detection limit on one decimal point: 2.26x10-7 for DPV and 2.30x10-7 M for SWV techniques in acetate buffer at pH 4.70. The repeatability of the methods was within 0.82-0.78% for peak potentials and 1.16-0.71% for peak currents. All necessary validation parameters were investigated as detailed in all media. The complexes have been screened for antibacterial activity and results were compared with the activity of the uncomplexed antibiotic against Pseudomonas aeruginosa, Kluvyeromyces fragilis, Saccharomyces cerevisiae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Bacillus megaterium, Candida albicans, Mycobacterium smegmatis, Bacillus cereus, Enterococcus cloacae and Micrococcus leteus. The copper complex was found to be more potent against two bacterial species than the uncomplexed CEF.

Keywords: Cefaclor, Copper complex, Electrochemistry, Spectral properties, Biological activity. 1. INTRODUCTION Almost all drugs that are currently in use are organic non metal substances. However, the discovery of antitumor properties of cisplatin, cis-[Pt(Cl)2(NH3)2], and its subsequent clinical success have prompted a vast number of investigation in metal complex area [1]. The best known metal complex used in the treatment of various cancerous malignancies is the platinum compound (cisplatin). Nowadays, cisplatin is one of the best-selling anti-cancer drugs worldwide. Its antitumor properties were found coincidentally. Transition-metal ions play a number of critical roles in biological and pharmaceutical sciences. The interactions of drug and metal ions have been thoroughly studied especially because of their interesting biological and chemical properties. Based on their wide spectrum of coordination numbers and geometries as well as kinetic properties, metal compounds enable unique mechanisms of drug action that can not be realized by organic agents. It is supposed that metal ions (especially copper II) are involved in antibacterial effectiveness of some agents. Many drugs possess modified toxicological and pharmacological properties in the form of metal complexes. The most widely studied metal in this respect is copper (II) *Address correspondence to this author at the Kahramanmaras Sutcu Imam University, Faculty of Science and Arts, Department of Chemistry, Campus of Avsar, 46100 Kahramanmaras, Turkey; Tel: 00 90 344 219 12 85; Fax: 00 90 344 219 10 42; E-mail: [email protected]

1573-4110/10 $55.00+.00

which has proved beneficial in diseases such as tuberculosis, gastric ulcer, rheumatoid arthritis and cancer [2]. These results encouraged us to investigate the coordination chemistry of antibiotics with transition metal ions in an attempt to examine the modes of binding in the solid state and to study their biological activity. Cephalosporins are referred to as the -lactam antibiotics, which are among the oldest and most frequently prescribed of naturally occurring antimicrobial agents. The key intermediate for semisynthetic production of a large number cephalosporins is 7-aminocephalosporanic acid, which formed by the hydrolysis of cephalosporin C produced by fermentation [3]. -lactam antibiotics easily interact with metal ions and this interaction is of a complex nature. This interaction is of importance as this may affect the drug absorption through the membranes. Metal ions are known to accelerate the rates of chemical reactions. Cephalosporins are structurally and pharmacologically related to the penicillin. Like penicillin, cephalosporins have a -lactam ring structure that interferes with the synthesis of the bacterial cell wall and so are bactericidal [4]. Currently, CEF is used to treat certain infections caused by bacteria such as pneumonia and ear, lung, skin, throat and urinary tract infections.

© 2010 Bentham Science Publishers Ltd.

Synthesis, Characterization, Biological Activity and Voltammetric Behavior

The determination of CEF is important not only in the field of human health for pharmacokinetic analysis but also for the quality control in food and fermentation industry to control the illegal use of it in food processing and preservation [5]. A wide variety of analytical methods have been reported for the determination of cephalosporins in their pure form, in pharmaceutical preparations and in biological fluids. These methods include spectrophotometry [6, 7], atomic absorption spectrometry [8], fluorometry [9, 10], polarography [11], high-performance liquid chromatography [12, 13], capillary electrophoresis [14] and chemiluminescence [15]. Electroanalytical methods have proved to be useful for the development of very sensitive and selective methods for the determination of organic molecules, including drugs and related molecules such as cephalosporins [16, 17] in dosage forms and biological fluids [18, 19]. Electrochemical techniques also help for the identification of the redox mechanism of drug compounds and provide important information about metal-drug complexes. The knowledge about the electrochemical properties of a drug is an important pharmaceutical tool since it can be relevant to understand its metabolic fate and pharmacological activity or its role in in vivo redox processes [20]. Surveying the literature revealed that no electrochemical method was reported for the determination of CEF except one indirect polarographic study related to Rodrigues et al. [21]. The study by Rodrigues et al. is related to the indirect polarographic and cathodic stripping voltammetric determination of CEF in an alkaline degradation product. However, nothing has been published concerning electrochemical behavior and voltammetric determination of CEF at solid electrode in pharmaceuticals. As a part of our continuous work, we reported the synthesis and characterization of several metal complexes that contain some drugs [22-27]. The aim of this study is the investigation of synthesis as well as spectral and electrochemical characterization and antimicrobial activity of CEF-metal complexes. In addition, we tried to demonstrate the interactions of CEF-metal complexes and develop a fast and rapid voltammetric determination of CEF via complexation in this study. In the presented work, copper (II) has been utilized for the determination of CEF. 2. EXPERIMENTAL PROCEDURE 2.1. Apparatus Elemental analyses (C, H, N) were performed using a LECO CHNS 932 elemental analyzer. IR spectra were obtained using KBr discs (4000-400 cm-1) on a Shimadzu 8300 FT-IR spectrophotometer. The electronic spectra in the 200900 nm range were obtained on a Perkin Elmer Lambda 45 spectrophotometer. Magnetic measurements were carried out by the Gouy method using Hg[Co(SCN)] as calibrant. Molar conductance of the metal complexes was determined in DMF (~10-3 M) at room temperature using a Jenway Model 4070 conductivity meter. Mass spectra of the ligands were recorded on a LC/MS APCI AGILENT 1100 MSD spectrophotometer. The metal contents of the complexes were determined by an Ati Unicam 929 Model AA Spectrometer in

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solutions prepared by decomposing the compounds in aqua regia and then subsequently digesting in concentrated HCl. The thermal analysis studies of the complexes were performed on a Perkin Elmer Pyris Diamond DTA/TG Thermal System under nitrogen atmosphere at a heating rate of 10oC/ min. All voltammetric measurements at the glassy carbon electrode were performed using a BAS 100W (Bioanalytical System, USA) electrochemical analyzer. A glassy carbon working electrode (BAS; : 3mm diameter), an Ag/AgCl reference electrode (BAS; 3M KCl) and platinum wire counter electrode and a standard one-compartment three electrode cell of 10mL capacity were used in all experiments. The glassy carbon electrode was polished manually with aqueous slurry of alumina powder (: 0.01 μm) on a damp smooth polishing cloth (BAS velvet polishing pad), before each measurement. All measurements were realized at room temperature. Mettler Toledo MP 220 pH meters was used for the pH measurements using a combined electrode (glass electrode reference electrode) with an accuracy of ±0.05 pH. DPV conditions were: pulse amplitude, 50 mV; pulse width, 50 ms; scan rate, 20 mVs1 and SWV conditions were: pulse amplitude, 25 mV; frequency, 15 Hz; potential step, 4mV. 2.2. Reagents CEF and its dosage forms (Losefar capsules) were kindly provided by Eczacıbaı Pharm. Comp. (Istanbul, Turkey). Metal salts (CuCl2·2H2O, CoCl2·6H2O and NiCl2·6H2O) were purchased from Merck and were used without further purification. All chemicals for preparation of buffers and supporting electrolytes such as H2SO4, H3PO4, NaH2PO4 , Na2HPO4, H3BO3, CH3 COOH, NaOH were reagent grade (Merck or Sigma). 2.3. Synthesis of Metal Complexes Copper (II), cobalt (II) and nickel (II) complexes (Scheme 1) were obtained according to a general procedure: A solution of the metal salt (1 mmol) in absolute MeOH (25 mL) was added to a solution of the CEF ligand (1 mmol) in absolute MeOH (20 mL) and the mixture was boiled under reflux for 6–7 h. At the end of the reaction, determined by TLC, the precipitate was filtered o, washed with distilled water and then EtOH, and dried under vacuum. O S NH NH2

N

Cl

O

+

MCl2 XH2O

C O HO

O S NH NH2

N

Cl

O Cl M: Cu(II), Co(II), Ni(II)

M

H2O

C O O

Scheme 1. The synthesis mechanism of solid complex of metal:CEF (1:1).

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[Cu(CEF)(H2O)(Cl)]: Yield: 70%. mp. 215 oC. Anal. calc. for C15H15Cl2CuN3O5S: C, 37.24, H, 3.12, N, 8.69, Cu, 13.13. Found: C, 37.17, H, 3.16, N, 8.62, Cu, 13.10. MS, m/z (positive ion): 487.5 [M+3]+. (-1cm2 mol-1): 23. eff (B.M.): 1.70. IR (KBr, cm-1): 3448 (OH), 3238 and 3054 (NH2), 1775 (C=O)amid, 1590 (M-OOC), 588 (M-O) and 501 (M-N). UV-Vis (max, nm): 530. [Co(CEF)(H2O)(Cl)]: Yield: 67%. mp. 233 oC. Anal. calc. for C15H15Cl2CoN3O5S: C, 37.60, H, 3.16, N, 8.77, Co, 12.30. Found: C, 37.64, H, 3.12, N, 7.71, Co, 12.35. MS, m/z (positive ion): 482.4 [M+3]+. (-1cm2 mol-1): 36. eff (B.M.): 4.20. IR (KBr, cm-1): 3423 (OH), 3269 and 3165 (NH2), 1757 (C=O)amid, 1590 (M-OOC), 517 (M-O) and 476 (M-N). UV-Vis (max, nm):428. [Ni(CEF)(H2O)(Cl)]: Yield: 62%. mp. 240 oC. Anal. calc. for C15H15Cl2N3NiO5S: C, 37.61, H, 3.16, N, 8.77, Ni, 12.25. Found: C, 37.60, H, 3.19, N, 8.75, Ni, 12.23. MS, m/z (positive ion): 482.1 [M+3]+. (-1cm2 mol-1): 17. eff (B.M.): 3.05. IR (KBr, cm-1): 3440 (OH), 3255 and 3058 (NH2), 1631 (C=O)amid, 1590 (M-OOC), 586 (M-O) and 503 (M-N). UV-Vis (max, nm): 497. 2.4. Determination of the Stoichiometric Ratio of the Reaction 2.4.1. Job’s Method The Job’s method of continuous variation was employed. Master equimolar (5103 M) acetonitrile:water mixture (1:1, v:v) solutions of CEF and metals (Cu(II), Co(II) and Ni(II)) were prepared. Series of 10 mL portions of the master solutions of CEF and metals were made up comprising different complementary proportions (0:10, 1:9, 9:1, 10:0, inclusive) in 10 mL calibrated flasks containing 1 mL of acetate buffer solution (pH 4.7). The solution was further manipulated as described under the general recommended procedures.

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completed to the volume with acetonitrile/water mixture. The content of the flask was sonicated for 10 min for complete dissolution. The sample from the clear supernatant liquor was withdrawn. One mL of CuCl2·2H2O was added and volume was completed to 10 mL with the selected supporting electrolyte. This solution was then transferred to a voltammetric cell and DP and SW voltammograms were recorded. The nominal content of CEF in each capsules were determined referring to the related regression equations. 2.6. Biological Activity The antibacterial activities of CEF and its synthesized metal (II) chelates were screened in vitro using the disc diffusion method [24, 26, 27]. The chosen strains, Pseudomonas aeruginosa, Kluvyeromyces fragilis A, Saccharomyces cerevisiae WET, Staphylococcus aureus, Escherichia coli ATCC, Klebsiella pneumoniae FMC, Bacillus megaterium DSM, Candida albicans, Mycobacterium smegmatis CCM, Bacillus cereus EU, Enterococcus cloacae ATCC and Micrococcus leteus LA were obtained from Microbiology Lab-oratory, Department of Biology, Faculty of Science and Arts, Kahramanmaras, Turkey. Test solutions of CEF and its Cu (II), Co(II) and Ni(II) complexes were prepared in DMSO. The bacteria were cultured for 24 h at 37 o C in an incubator. Muller Hinton broth was used for preparing basal media for the bioassay of the organisms. Nutrient agar was poured onto a plate and allowed to solidity. The test compounds solutions (DMSO) were added drop wise to a 10 mm diameter filter paper disc plates at the centre of each agar plate. The plates were then kept at 5 oC for 1 h and transferred to an incubator maintained at 37 oC. The width of the growth inhibition zone around the disc was measured after 24 h incubation. 2.7. Electrochemical Procedures 2.7.1. Reagents

2.5. Spectrophotometric Determination of CEF 2.5.1. Procedure for Calibration Graph A stock solution containing 1x10-3 M of CEF was prepared in acetonitrile:water mixture (1:1, v:v) and was further diluted as appropriate. This solution was stable at least for a day if kept in the refrigerator. CuCl2·2H2O solution 1x10-3 M was prepared in same media. Acetate buffer (pH 4.7) solution was prepared by mixing specific volumes of acetic acid (0.1 M) and sodium acetate (0.1 M). For calibration curve, aliquot volumes of the stock were transferred to the solution containing 1x10-5 – 2x10-4 M of CEF into a series of 10 mL volumetric flasks. One mL of CuCl2·2H2O was added to each solution and completed to the volume with the selected buffer. The absorbance was measured at 530 nm against a blank. Calibration curve was constructed and the regression equation was derived. 2.5.2. Procedure for Capsules Ten capsules (each capsules contains 250 mg CEF) were accurately weighed, emptied and pulverized by pestle in a mortar. An adequate amount of this powder, corresponding to a stock solution of a concentration of 1x10–3 M of CEF, was weighed, transferred into a 100mL-calibrated flask and

Both stock solution of CEF–Metal (II) complexes (as described in section 2.3) and in solution complex (as described in section 2.5) were used for electrochemical studies. Complex working solutions under voltammetric investigations were prepared by dilution of the stock solution. Dissolved oxygen was removed by passing pure nitrogen through the solution for 3.0 min prior to electrochemical analyses. Two different supporting electrolytes, acetate buffer (0.2M; pH 3.7 –5.7) and phosphate buffer (0.2M; pH 3.0– 8.0), were prepared in doubly distilled water. The calibration curve for DPV and SWV analysis was constructed by plotting the peak current against the CEF–Cu complex in solution media. Linearity range were between 2x10-6 and 6x10-5 M for DPV and 4x10-6 and 6x10-5 M for SWV. 2.7.2. Capsule Procedure The procedure was followed as described in section 2.5.2. Analyzed solutions were prepared by taking aliquots of the clear supernatant and diluting it with the selected supporting electrolyte. Voltammograms were recorded accord-

Synthesis, Characterization, Biological Activity and Voltammetric Behavior

ing to the DPV and SWV parameters and as in solution CFCu complex. 2.7.3. Recovery Experiments In order to exclude interferences by excipients in capsule dosage form, known amounts of the pure drug were added to different pre-analyzed formulations of CEF-Cu in solution complexes and the mixtures were analyzed by the proposed techniques. After five repeated experiments, the recovery results were calculated using the calibration equation. 3. RESULTS AND DISCUSSION The structures of the new complexes reported herein were established with the help of their UV-Vis, IR, mass and all micro-analytical data. The stoichiometry of the reaction between CEF and metals were investigated by Job’s method. Job’s plot for M+2 complex is presented in Fig. (1). Job’s plots revealed an Xmax=1/2 of for [Cu(CEF)(H2O)(Cl)], [Co(CEF)(H2O)(Cl)], and [Cu(CEF)(H2O)(Cl)] complexes. Hence, the stoichiometry of the metal-ligand complexes of CEF was 1:1 at used concentration of the ligand (5103 M). The elemental analyses data agree well with the proposed formulate for the complexes. Copper (II), cobalt (II) and nickel (II) complexes were dark brown, brown and pale brown, respectively. All metal complexes were stable in air.

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stretching frequencies, respectively. These bands have appeared in the spectra of all complexes around the same wave number. This type of vibration showed that –NH2 and –NH groups did not participate to coordination with metal. The lactam (C=O) band appears at 1749 cm-1. The complexes that have a 1:1 molar ratio of metal: CEF did not change amide or -lactam bands. These observations suggest that CEF is not coordinated to the metal ions through amide or lactam oxygen. The carboxylate ligand can bind to a metal atom as a mono-dentate ligand, causing changes in the relative positions of the asymmetric and symmetric vibrations [28]. The spectrum of CEF exhibits a band at 1500 cm-1 belongs to carboxylate group. A new band at 1590 cm-1 observed in the IR spectra of complexes suggested [29] bonding of the carboxylate the (M-OOC) group. The presence of the strong (M-N) and (M-O) stretching vibration at 476503 cm-1 and 517-588 cm-1 for the metal complexes (absent in CEF), support coordination of CEF through nitrogen of lactam ring and carboxylate. Moreover, the -lactam band at 1749 cm-1 moved to lower frequency by 10-15 cm-1 indicating involvement /coordinating of the -lactam oxygen to the metal ion. In the present studies, the CEF molecule has several potential donor atoms. However, the molecule can provide a maximum two donor sites to the metal atom due to steric constrains. The assumption that the CEF molecule coordinates through the carboxylate and -lactam nitrogen seems likely from molecular modeling studies. In the complexes, [M(CEF)(H2O)(Cl)] where M: Cu(II), Co(II) and Ni(II), CEF acts as bi-dentate and hence forms a tetrahedral geometry. 3.2. Electronic Properties The electronic spectra of complexes of CEF were measured in DMSO in order to obviate the effect of the solvent. The UV-Vis spectra of CEF and its Cu(II), Co(II) and Ni(II) complexes present two absorption maxima at 265 and 301 nm assigned to -* and n-* transitions within the organic ligand. The spectra of the complexes contain same absorption bands in the range 588-428 nm (relatively weak, lowenergy bands), which may be assigned to the d-d* transition in a tetrahedral configuration. This data are in accordance with the assumption for the formation of M-N and M-O bands [30, 31].

Fig. (1). The Job curves of [Cu(CEF)(H2O)(Cl)] (); [Co(CEF)(H2O)(Cl)] ( ) and [Ni(CEF)(H2O)(Cl)] ( ) complexes.

The synthesized complexes can only be solved in DMSO, DMF and acetonitrile:water (1:1; v:v) mixture. They are insoluble in ethanol, methanol or 1.4-dioxane. The conductivity values, measured in DMSO at room temperature, fall within the range 17-36 ohm-1cm-2mol-1 suggesting the [M(CEF)(H2O)(Cl)] complex to be all electrolytic in nature. Efforts to grow good crystals for X-ray diffraction studies were unsuccessful due to their poor solubility in common organic solvents. 3.1. IR Spectra The spectra of the free CEF showed absorption bands at 3295 and 3050 cm-1 corresponding to (NH2) and (NH)

3.3. Magnetic Properties The magnetic moment of the complexes was measured at room temperature. On the basis of the magnetic and spectral evidence, the metal complexes have mononuclear structures in which the metal cations have an approximately tetrahedral environment [31]. The μeff values have been found 1.70, 4.20 and 3.05 for [Cu(CEF)(H2O)(Cl)], [Co(CEF)(H2O)(Cl)], and [Cu(CEF)(H2O)(Cl)], respectively. 3.4. Atomic Absorption Spectra The ratios of the metal present in all three complexes were determined by atomic absorption spectroscopy. The complexes were decomposed in HNO3/H2O2 (1/1) and then dissolved in 1.5 N HNO3. The amounts of the metals were determined. They support the structures given in the Scheme 1.

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3.5. Mass Spectra As seen in Scheme 1, there are free nitrogen, oxygen, and sulfur atoms in all complexes. These atoms may be binding the hydrogen atoms. Therefore, the molecular ion peaks of the complexes were not shown in mass spectra. Indeed, the molecular ion peaks of [M+3]+ were shown at m/z 487.5, 482,4 and 482.1 for Cu(II), Co(II) and Ni(II) complexes, respectively. The mass spectrum of [Cu(CEF)(H2O)(Cl)] was given in Fig. (2).

Fig. (2). The mass spectrum of [Cu(CEF)(H2O)(Cl)] complex.

3.6. Thermal Analyses Thermogravimetric analysis is a useful technique for the determination of the thermal stability and structural elucidation of various insoluble and infusible compounds [32], but

Fig. (3). Thermal analysis curves of the [Cu(CEF)(H2O)(Cl)] complex.

Tarinc et al.

to date, only a limited number of reports concerning thermal data and solution thermochemistry of metal-drug complexes have been appeared in the literature. In this study, the thermal behavior of the complexes was characterized using DTA and TGA/DTG methods. The DTA/TG measurements of the complexes were carried out in the 55-1250 oC range. The complexes contain the coordinated chloride ion and water molecules. There is one route in removal of the coordinated water molecules in the 116-219 oC temperature range from the complexes. This mass loss equals the 3.19-4.15 of the total. Moreover, the coordinated chloride ion losses from complexes in the 286-316 oC temperature range (wt. loss 6.06.53%). At higher temperatures (400-1200 oC), all complexes decompose to give the approximate metal oxide. The thermal curves of the [Cu(CEF)(H2O)(Cl)] were given in the Fig. (3). The lower stability of the Co(II) complex might be attributed to the autocatalytic effect of the Co(II) ion. In addition, intermolecular factors may operate and control the stability of the complexes. The factors affecting the thermal stability of transition metal complexes in the solid state are not well understood. There are many examples in which the following order of thermal stability was established; Mn > Fe > Co > Ni > Cu > Zn. This is the reverse of the Irwing-Williams series, i.e. the stability order of the complexes in aqueous solution. This behavior can be explained by a decrease of the intermolecular forces in the solid state with increasing strength of the intra-molecular metal ligand bonds. In the complexes, the following thermal stability order can be recognized; Ni > Cu > Co. 3.7. Microbiological Screening The susceptibility of certain strains of bacterium towards CEF and its metal complexes was judged by measuring the size of inhibition zone diameter. Antibacterial activities of CEF and the complexes have been carried out with six gram

Synthesis, Characterization, Biological Activity and Voltammetric Behavior

Current Analytical Chemistry, 2010, Vol. 6, No. 4

positive [Staphylococcus aureus, Bacillus megaterium, Mycobacterium smegmatis, Bacillus cereus, Enterococcus cloacae and Micrococcus leteus] and six gram negative [Pseudomonas aeruginosa, Kluvyeromyces fragilis, Saccharomyces cerevisiae, Escherichia coli, Klebsiella pneumoniae, Candida albicans] bacteria. The test solutions were prepared in DMSO. The results of the antibacterial activities are summarized in Table 1. The synthesized compounds were found to have remarkable bactericidal and fungicidal properties. It is, however, interesting that the biological activity was enhanced with complexation with the metal ion. From the structure point of view, it is clear that formation of the chelate derivatives in the 1:1 molar ratio (M:L) sometimes increase the biological activity as appeared from the CEF-copper (II) complex. Especially, while CEF did not show any antibacterial effect against the Klebsiella pneumoniae and Bacillus megaterium, [Cu(CEF)(H2O)(Cl)] complex have antibacterial effects against these bacteria. Inhibition zones were 18 and 15 for Klebsiella pneumoniae and Bacillus megaterium, respectively. Moreover, enhanced activities of the derivatives of metal complexes compared to the free ligand may be due to the chloride ion around the central metal ion arising from chelation in 1:1 molar ratio (M:L). Such an increased activity of the metal chelates as compared to the CEF can be explained on the basis of chelation theory [33]. Chelation considerably reduces the polarity of the metal ion because of the partial sharing of its positive charge with the donor groups and possible p-electron delocalization over the chelate ring. Such chelation could increase the lipophilic character of the central metal atom, which subsequently favors the permeation through the lipid layer of cell membrane. The mode of action of the complexes may involve the formation of the hydrogen bond through the primer and secondary amines, thiol, -lactam oxygen and free carbonyl oxygen groups with the active centers of the cell constituents resulting in the interference with normal cell process.

Table 1.

321

3.8. Electrochemical Studies No previous electrochemical data were available concerning the solid electrode behavior of CEF. Actually, the electrooxidative response of CEF is occurred on high positive potentials as an ill-defined wave. Cyclic voltammetry (CV), differential pulse (DPV) and square wave (SWV) voltammetric techniques have been utilized to elucidate and confirmation the possible complex formation reaction between CEF and metal ions such as Cu(II), Co(II), and Ni(II) on glassy carbon electrode. Fig. (4) shows comparative CV of CEF in the presence of these metal ions at both anodic and cathodic directions for: (1) CEF alone, (2) metal ions alone, (3) metal ions – CEF in solid complex. Evidence of a complex formation is established as a result of the increase of the copper peak currents at about + 0.0027 V and + 0.20 V on anodic direction and -0.09 V and +0.23 V on cathodic direction such as the case of copper-CEF complexes (Fig. (4a and 4d)). Fig. (4a) showed that copper retains its well-defined peak and the increase in the diffusion current was more than it was with the other metal ions. CEF and metal ions were studied at both anodic and cathodic directions. As a result of the working with different metal ions, we decided to work with copper complex for it gave the best response, peak shape and reproducible results in electroanalytical studies. Electroanalytical results on complexation studies were in accordance with spectrophotometric method. Cobalt and Nickel complexes did not give reliable results for measuring at both directions (Fig. (4b, c, e and f)). The voltammograms of CEF alone in a wide potential range showed one illdefined wave peak at +1.3 V with oxidation process. This uncertain wave is not convenient for analytical measurements at a glassy carbon electrode (Fig. (4)). The better peak shape, reproducible and sensitive results and partial or total lack of interferences were obtained on anodic direction of copper (II):CEF solid synthesized complex. The copper (II) ion yields a quasi-reversible redox couple, located at +0.011

Antimicrobial Activity Data for the CEF and its Metal Complexes Compound

Gram Positive Bacteria

Gram Negative Bacteria Diameter of Inhibition Zones (mm)a

1

3

4

5

6

1'

2'

3'

4'

5'

6'

a

34

30

38

46

45

45

42

CEF

46

35

45

-

-

[Cu(CEF)(H 2O)(Cl)]

21

31

36

18

15

33

26

29

33

36

34

36

[Co(CEF)(H 2O)(Cl)]

7

11

7

8

7

-

-

-

10

-

13

-

[Ni(CEF)(H2O)(Cl)]

11

-

-

-

15

-

12

14

10

-

-

7

P10

36

38

33

29

21

22

8

-

16

3

-

-

AMP20

26

24

31

30

20

28

10

-

9

23

12

-

41

40

39

33

34

54

-

49

30

10

-

CEF30 a

2

8

7

35 8

7

a

a

*: The X values is in the range 7.6x10 – 8.3x10 for the gram positive bacteria and 8.7x10 – 9.6x10 for the gram negative bacteria (X : CFU /mL (inoculum)) 1: Staphylococcus aureus, 2: Bacillus megaterium, 3: Mycobacterium smegmatis, 4: Bacillus cereus, 5: Enterococcus cloacae and 6: Micrococcus leteus, 1': Pseudomonas aeruginosa, 2': Kluvyeromyces fragilis, 3': Saccharomyces cerevisiae, 4': Escherichia coli, 5': Klebsiella pneumoniae, 6': Candida albicans. P10, Penicillin G 10 μg/disc; AMP20, Ampicillin 10 μg/disc; CEF30, Cefotaxime 10 μg/disc. - : No inhibition zone is determined

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Fig. (4). Cyclic voltammograms for (1:1) complexes; (A) Cu: CEF; (B) Co: CEF; (C) Ni: CEF; on the anodic direction and (D) Cu: CEF; (E) Co: CEF; (F) Ni: CEF complexes on the cathodic direction in acetate buffer at pH 4.70. (1) 1x10-4M CEF; (2) 1x10-4M Metal (II) and (3) 1:1 solid synthesized complexes between metal (II) and CEF.

V and -0.055 V, in Fig. (4a). As a result of these experiments, we decided to continue with oxidation direction for further studies. CEF-copper complex was utilized for sensitive detection of CEF.

As seen in Fig. (5a and b), at both directions, CEF by itself does not have any measurable wave, Cu has a peak at about -0.061V at anodic direction and +0.098 V at cathodic direction. CEF-Cu(II) solid synthesized complex has one

Synthesis, Characterization, Biological Activity and Voltammetric Behavior

sharp and well defined peak at about -0.054 V at oxidation direction and +0.107 V at reduction direction (Fig. (5)). It is worthy to mention that, in agreement with these results and according to DPV curves (Fig. (5)), evidence of 1:1 complex formation of Cu(II): CEF is investigated for the sensitive and selective electroanalytical determination of CEF at anodic direction. As indicated from Figs. (4 and 5), there is an evidence of a complex formation reaction between CEF and Cu(II). When copper (II):CEF was occurred, the potential of copper was shifted to more positive potentials and the current was increased in solution using both media and solid synthesized complexes (Fig. (6)). Complex formation is obvious not only from the positive shift in oxidation potentials but also the appearance of a new peak at about +0.196 V with solid synthesis complex. Also, confirmation of the interaction CEF-Cu(II) complex is established as a result of the increasing copper peak current on solid synthesis complex and decreasing of the copper peak current in solution (Fig. (6)).

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various situations. The peak current of the synthesized solid complex showed a sharp peak and the best reproducible results occurred between -400 mV and +400 mV at the anodic direction for analytical applications (Fig. (7)).

Fig. (6). Cyclic voltammograms of 1x10-5M CEF alone (1); 1x10M Cu(II) alone (2); solid synthesized (1:1) complex of Cu(II): CEF (3) and in solution complex of (1:1) Cu (II) : CEF on the anodic direction. 5

At the beginning of this study, CEF was investigated by cyclic and linear sweep voltammetric studies with the aim of characterizing its electrochemical behavior on a glassy carbon electrode. After realizing these characterization studies, CEF was subjected to a voltammetric determination in the DPV and SWV modes. For CEF, the effect of pH was investigated between a pH range of 3.00 and 8.00, where complex was stable using acetate (0.2M; pH 3.7 –5.7) and phosphate (0.2M; pH 3.0–8.0) buffers by CV method. CV is perhaps the most effective and versatile electroanalytical technique available for the mechanistic study of redox system [34, 35]. The pH of the supporting electrolyte has a significant effect on the electrooxidation of CEF at the glassy carbon electrode. CV voltammograms of CEF:Cu(II) solid synthesized complexes exhibited one well-defined peak. The peak developed best and became sharper in acetate buffer at pH 4.70.

Fig. (5). Differential pulse voltammograms of 1x10-5M CEF alone (1); 1x10-5M Cu (II) alone (2) and 1:1 solid synthesized complex of Cu (II): CEF (3) on the anodic direction (a) and cathodic direction (b).

Dependence of peak currents and potentials was established by switching of the initial and high potentials using DPV and SWV techniques. The SWV curves were shown in Fig. (7) for representing the effect of the applied potential. With changing initial potential, the copper complex showed

Plots of pH versus Ep and ip have been investigated using CV techniques. The peak potential of the anodic process moved to less positive potential values by raising the pH. The plot of the peak potential (Ep) versus pH showed one straight line between pH 3.00 and 8.00, which can be expressed by the following equations in both acetate and phosphate buffers: Ep (mV) = 372.47 –28.79pH; r: 0.989 The effect of pH on peak current of CEF in the range of pH 3.00 –8.00 was also evaluated. The best and sharpest peak and reproducible results were obtained in pH 4.70.

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Tarinc et al.

Fig. (7). Initial potential effect on the solid synthesized complex of Cu(II): CEF. (1) 1x10-5 M Cu(II) alone and (2) 1:1 solid synthesized complex. The initial and final potentials of square wave voltammograms: (A) Initial potentials: -0.75 V; Final potentials: 0.75 V. (B) Initial potentials: -0.60V; Final potentials: 0.60V. (C) Initial potentials: -0.50V; Final potentials: 0.50V. (D) Initial potentials:-0.40V; Final potentials: 0.40V. (E) Initial potentials: -0.30V; Final potentials: 0.30V.

acetate buffers. Therefore this media was chosen in this study as the supporting electrolyte for the electroanalytical part. The dependence of peak currents and peak potentials on the scan rate (v) was studied in the range of 10 –1000 mVs–1. Scan rate studies showed that the anodic process on glassy carbon electrode is under diffusion or adsorption control. The peak potential was shifted to less positive values at an increasing scan rate. A linear relationship was observed be-

tween peak current and square root of the scan rate corresponding to the equation: ip (A) = 0.10v1/2 (mVs–1) - 0.24 r: 0.994 A plot of logarithm of peak current (log I) versus logarithm of scan rate (log v) gave a straight line within the same range. The linear relationship was obtained as follows: Log ip (A) = 0.58logv(mVs–1) – 1.30 r: 0.997

Synthesis, Characterization, Biological Activity and Voltammetric Behavior

The slope of 0.58 is close to the theoretically expected value of 0.5 for an ideal reaction at solution species and a purely diffusion–controlled current [35, 36]. In order to improve voltammetric methods for quantitative analysis of the CEF, DPV and SWV techniques were selected. DPV and SWV techniques are effective and rapid electroanalytical techniques with well-established advantages, including good discrimination against background currents and low detection limits [34, 35]. Based on the above study, the best condition for analytical applications proved to be an acetate buffer of pH 4.70.

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The results of spectrophotometric and voltammetric techniques were compared with each other in Table 3. The amounts of CEF are fairly close to the labeled amounts in both techniques. However, the electroanalytical methods are sensitive, selective and more precise than the spectrophotometric analysis. The student-t and F tests were carried out on the data and statistically examined the validity of the re-

Interactions between CEF and Cu(II) complex was utilized for sensitive detection of CEF up to μM concentrations. Before this complexation, CEF could not be determined alone using any electroanalytical methods. The quantitative assessment is based on the dependence of the peak current on the frequent respective concentration of CEF: Cu(II) complex was found linear within a concentration range of 1x10–6 to 3x10–5M by using both DPV and SWV techniques in solid synthesis complex. Also, in solution CEF: Cu(II) complex concentration ranges were 2x10–6 to 7x10–5M for DPV; 4x10–6 to 7x10–5M for SWV. These results indicated that the response was diffusion-controlled within these ranges. Typical DPV curves of solid synthesis complex and typical SWV curves of in solution complex, were shown in Fig. (8a and b), respectively. The developed techniques were validated and the results are summarized in Table 2. The precision of the method was investigated by repeatedly (n=5) measuring peak potential and peak current of CEF: Cu(II) complex responses for within a day (repeatability) and over three consecutive days (reproducibility) for both techniques These results were shown as the RSD% values in Table 2. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as (3 /m) and (10 /m), respectively, where  is the standard deviation of response (three runs) and m the slope of the calibration curve. LOD and LOQ values confirmed the sensitivity of the proposed method, were shown in Table 2. The low values of SE of slope, the intercept and a correlation coefficient greater than 0.999 in supporting electrolyte confirmed the precision of the proposed method. The proposed DP and SW voltammetric techniques were applied to the assay of CEF in Losefar capsules in solution complex with CEF: Cu(II), using the related calibration straight lines without any sample extraction, evaporation or filtration and after adequate dilutions. The samples were used after adequate dilutions. The accuracy of the proposed method was determined by its recovery during spiked experiments. Recovery studies were carried out after addition of known amounts of pure drug to the pre-analyzed formulations of CEF using in solution complexation technique. In order to obtain the possible interactions from the excipients, the standard addition technique was applied to the same preparations, which were analyzed using the related calibration curve. Recovery experiments using the developed assay procedure further indicated the absence of interference from commonly encountered pharmaceutical excipients used in the selected formulations Table 3. The results indicate the validity of proposed techniques for the determination of CEF in capsules Table 3.

Fig. (8). DPV curves for solid synthesized complex (a) and SWV curves for in solution complex (b); Supporting electrolyte (1); 1x105 M (2); 2x10-5 M (3); 3x10-5 M (4) CEF:Cu (II) complexes in acetate buffer at pH 4.70.

sults by spectrophotometric and electroanalytical methods. At the 95% of the confidence level, the values of t- and Ftests (calculated from the experiments) were less than that of theoretical t- and F-values showing that there are no significant differences between the electroanalytical and spectrophotometric methods with regard to accuracy and precision. However, the proposed electroanalytical methods could be successfully applied for CEF assay analysis in capsule dosage form without any interference. As confirmed by student’s t test, the calculated t value did not exceed the theoretical value for significance level of 0.05. There was no significant difference between the performance of the two techniques with regards to applicability and simplicity.

326 Current Analytical Chemistry, 2010, Vol. 6, No. 4

Table 2.

Tarinc et al.

Statistical Data for the Calibration Graphs of CEF-Cu Complex by Voltammetric and Spectrophotometric Methods Spectrophotometric Method

Voltammetric Methods Solid Complex

Solution Complex Solution Complex

DPV Measured potential (V) and absorbance (nm)

SWV

0.13

DPV

0.14

-6

-5

0.13

-6

1.41x104

2.01x104

4.15x103

-1.08x10-2

-3.73x10-2

-6.84x10-2

0.1961

0.999

0.998

0.999

0.999

1x10 – 3x10

Slope

4.51x104

4.86x104

Intercept

-5.81x10-2

Correlation coefficient

0.999

6.72x10

-6

2x10 – 7x10

2

3.41x10

2

-5

530 1x10 – 2x10-4

1x10 – 3x10

-5

0.20 4x10 – 7x10-5

Linearity range (M)

2

SWV

-6

3.49x10

-5

2

9.64x101

SE of slope

5.07x10

SE of intercept

6.99x10-3

9.27x10-3

1.28x10-2

1.26x10-2

1.05x10-2

LOD(M)

2.26x10-7

2.30x10-7

3.01x10-7

8.10x10-7

1.28x10-6

LOQ(M)

7.55x10-7

7.66x10-7

1.00x10-6

2.70x10-6

4.09x10-6

Repeatability of peak potential/wavelenght (RSD %)*

0.82

0.78

1.32

0.71

0.752

Repeatability of peak current/absorbance (RSD %)*

1.16

0.71

1.43

0.88

0.01

Reproducibility of peak potential/wavelenght (RSD%)*

1.33

2.15

2.66

2.10

0.02

Reproducibility of peak current/absorbance (RSD%)*

1.27

1.62

1.36

1.30

1.73

* Each value is obtained from five experiments

Table 3.

Results of the Assay and the Recovery Analysis of CEF-Cu Complex in Capsül Dosage Forms in Only Solution Complex by Voltammetric and Spectrophotometric Methods Voltammetric Methods

a

Spectrophotometric

DPV

SWV

Method

Labeled claim (mg per tablets)

500.00

500.00

500.00

Amount found (mg)a

501.38

502.52

501.25

RSD %

1.42

0.61

0.92

Bias %

-0.28

-0.50

-0.25

t-test

0.40

0.39

ttheoretical =2.31

F-test

0.32

0.59

Ftheoretical = 2.60

Added (mg)

40.00

40.00

40.00

Founda

40.16

40.24

40.29

Recovery %

100.41

100.60

99.59

Bias %

-0.41

-0.60

0.41

RSD % of Recovery

1.58

1.43

1.77

Mean value of the five determination.

To prove the absence of interferences by excipients, recovery studies were also carried out for all techniques. The accuracy of the analysis of all methods was determined by calculating the percentage relative error (Bias %) between

the measured and actual concentrations. The precision value around the mean value should not exceed 5% of the RSD % Table 3 [37-40].

Synthesis, Characterization, Biological Activity and Voltammetric Behavior

CONCLUSION This study described the synthesis, spectrophotometric and electrochemical characterization of CEF-metal complexes. This work contributes to a better elucidation of the complexation of CEF with Cu(II) ions. The complete electrochemical and spectrophotometric studies described in this paper demonstrated the complex formation circumstances for obtaining solid synthesized and in solution complexes. The spectral results indicate the complexation cites. The study also revealed the redox behavior of CEF:Cu(II) as well as pH, scan rate, solvent mixture and initial and final potential effects of the complex. The possible complex formation reaction that may occur between metal ions and CEF under investigation may indicate a possible effect of administrating this antibiotic with multivitamins that included trace elements. Possible interactions between metal and CEF as a complex were explained with the help of UV-Vis, IR, mass and electrochemical techniques. On glassy carbon electrode, an increase in the copper peak current on solid synthesis complex and occurring additional peak to the copper peak was the clear confirmation for interaction between Cu(II) and CEF. A sensitive, selective and rapid detection and determination of CEF was demonstrated based on the synthesized 1:1 ratio of CEF:Cu(II) solid and occurred in solution complexes. The proposed determination methods are simple, practical, sensitive, rapid and accurate. The complex of CEF with Cu(II) allows the electroanalytical determination of CEF with no interferences from neither excess reagent nor from the inactive ingredients in pharmaceutical dosage forms. Therefore, the proposed analytical techniques seem to be a useful tool for the determination of CEF in pharmaceutical dosage forms. Also these techniques show the potential to further develop these electroanalytical methods as a quick and low cost sensor for the detection of CEF in various media as well as biological samples.

Current Analytical Chemistry, 2010, Vol. 6, No. 4 [9]

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ACKNOWLEDGEMENT This research was supported by a grant TUBITAK (Grand No: 105T371) for Assoc. Prof. Dr. Aysegul Golcu.

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Received: January 12, 2010

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Revised: March 08, 2010

Accepted: March 25, 2010

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