A simple method for the spectrophotometric determination of cephalosporins in pharmaceuticals using variamine blue

EQ06-AF 26/6/08 11:45 Page 41 www.scielo.br/eq www.ecletica.iq.unesp.br Volume 33, número 2, 2008 A simple method for the spectrophotometric determination of cephalosporins in pharmaceuticals using variamine blue C. Pasha and B. Narayana* Department of Post Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangothri – 574 199, Karnataka, India. * Abstract: A simple spectrophotometric method for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin with variamine blue is presented. The determination is based on the hydrolysis of β-lactam ring of cephalosporins with sodium hydroxide which subsequently reacts with iodate to liberate iodine in acidic medium. The liberated iodine oxidizes variamine blue to violet colored species of maximum absorption at 556 nm. The absorbance is measured within the pH range of 4.04.2. Beer’s law is obeyed in the range of 0.5-5.8 µg mL–1, 0.2-7.0 µg mL–1, 0.2-5.0 µg mL–1 and 0.58.5 µg mL–1 for cefotaxime, ceftriaxone, cefadroxil and cephalexin respectively. The analytical parameters were optimized and the method is successfully applied for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin in pharmaceuticals. Keywords: cephalosporins determination; spectrophotometry; variamine blue. Introduction Cephalosporins structurally differ from pencillins by the heterocyclic ring system. Cephalosporins are pencillinase-resistant antibiotics with significant activity against both gram-positive and gram-negative bacteria. The key intermediate for semisynthetic production of a large number of cephalosporins is 7-aminocephalosporanic acid, which is formed by hydrolysis of cephalosporin C produced by fermentation [1]. A few thousand semisynthetic cephalosporins have been described in the scientific literature, but only a small number of these has shown clinical importance. Cefotaxime, ceftriaxone, cefradine and cephalexin are β-lactam antibiotics possessing a broad spectrum of antibacterial properties [2,3]. Several methods have been reported for the quantitative determination of cephalosporins. These Ecl. Quím., São Paulo, 33(2): 41-46, 2008 include fluorimetric [4], polarographic [5] and isotachophoretic methods [6]. Some reported spectrophotometric methods for the determination of these analytes are chloranilic acid [3], paramolybdate anion [7], molybdophosphoric acid [8] formation of a complex with Cu(II) [9], a reaction with potassium iodate in acidic medium [10]. Cefotaxime, ceftriaxone and cefradine were also determined in pharmaceuticals preparations [11-15], Urine [14,16-19] and human serum [20]. Recently a rapid development of chromatographic determination methods of pharmaceuticals has been observed too [21,22]. The hydrolysis of ß-lactum ring, which is the common feature for cephalosporins and pencillins, has been achieved by the sodium hydroxide addition. Major difficulties in the determination of cephalosporin have been encountered at the β-lactum ring hydrolysis step [23]. A β-lactum enzyme 41 EQ06-AF 26/6/08 11:45 Page 42 [24] has been used for the hydrolysed product of the analyte reacts with iodate in acid medium and liberates iodine. The liberated iodine oxidizes variamine blue to the violet colored species is the basis for the spectrophotometric determination of the analytes. The reaction mechanism followed the course similar to the one described for pencillins [25]. The proposed method has been successfully applied for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin in pharmaceuticals. Table 1. Structures of the cephalosporins studied. Experimental Apparatus A Systronics 2201 UV-VIS Double Beam Spectrophotometer with 1 cm quartz cell was used for the absorbance measurements and a WTW pH 330, pH meter was used. Reagents and solutions All chemicals used were of analytical grade and doubly distilled water was used for dilution of the reagents and samples. Cefotaxime, ceftriaxone, cefadroxil, and cephalexin stock solutions (1000 µg mL–1) were prepared by dissolving standard sodium cefotaxime (Alkem Lab. Ltd. Mumbai) or sodium ceftriaxone (Aristo Pharmaceuticals Ltd. Mumbai) or standard cefadroxil (Alkem Lab. Ltd. Mumbai) or standard cephalexin (Ranbaxy, India) in water. These compounds chosen to represent cephalosporins. They were prepared freshly, as required, by dissolving an appropriate amount of each antibiotic in water to provide a 1 µg mL–1 solution. The standard solution must be protected from contact with light. The structures of the cephalosporins studied are listed in table 1. Sodium hydroxide 0.1 mol L–1 aqueous solution, hydrochloric acid (Merck Limited, Mumbai) 1 mol L–1 aqueous solution, potassium iodate (S.D. fine – Chem Limited, Mumbai) 0.1 mol L–1 aqueous solution were used through out. Taxim (Alkem Lab. Ltd. Mumbai), Monocef (Aristo Pharmaceuticals Ltd. Mumbai), Cefadrox (Aristo Pharmaceuticals Ltd. Mumbai) and Sporidex (Ranbaxy, India) were examined. A 0.05% solution of Variamine Blue (E-Merck Limited, Mumbai) in (75: 25) water-ethanol mixture was used and stored in an amber bottle. 42 Procedure An aliquot of a sample solution containing 0.5 – 5.8 µg mL–1 of cefotaxime, 0.2-7.0 µg mL –1 of ceftriaxone, 0.2-5.0 µg mL –1 of cefadroxil and 0.5-8.5 µg mL–1 of cephalexin was transferred into a series of 25 mL calibrated flasks, 1 mL of 0.1 mol L–1 sodium hydroxide were added and the mixture was kept on a water bath (80°C) for 10 min. after being cooled to room temperature (27 ± 2°C), 1.5 mL of 0.1 mol L–1 potassium iodate and 2 mL of 1 mol L–1 hydrochloric acid were added The mixture was gently shaken until the appearance of yellow color, indicating the liberation of iodine, 1 mL of 0.05 % of variamine blue was then added to it followed by the addition 2 mL of 1 mol L–1 of acetate buffer of pH 4 and the reaction mixture was shaken for 2 min. The contents were diluted up to 25 mL with distilled water and mixed well. The absorbance of the oxidized species variamine blue formed was then measured at 556 nm against the reagent blank prepared in the same manner, without the analyte. The amount of the cefotaxime, ceftriaxone, cefadroxil and cephalexin present in the volume taken was computed from the calibration graph. Ecl. Quím., São Paulo, 33(2): 41-46, 2008 EQ06-AF 26/6/08 11:45 Page 43 Analysis of injection solution An appropriate amount of each antibiotic was dissolved in water so as to prepare 1mg mL–1 solution and then the recommended procedure was followed without modification. The presence of other substances caused no significant interference with the determination of antibiotics. Analysis of formulations Weighed an amount of the sample equivalent to about 250 mg cephalosporin and was dissolve in a sufficient amount of distilled water. The solution was shaken and filtered off through Whatman No. 1 filter paper and washed with water. The filtrate was diluted up to the mark with distilled water and made upto 100 mL (...truncated)