ISSN: 0973-494; CODEN ECJHAO E- Chemistry http://www.ejchem.net 2012, 9(4), 221-227 Validated Spectrophotometric Assay of Cefepime Hydrochloride and Cefuroxime Sodium Using a Tetrazolium Salt MARWA S. ELAZAZY * AND ABDALLA SHALABY Analytical Chemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt marwaelazazy78@yahoo.com Received 29 August 2011; Accepted 28 October 2011 Abstract: A simple, rapid and sensitive spectrophotometric method for the determination of micro amounts of cefepime hydrochloride and cefuroxime sodium is described. The method is based on reduction of 2,3,-triphenyltetrazolium chloride (TTC) by the cited drugs in slightly alkaline medium leading to formation of a highly colored formazan derivative. Different variables affecting the color development were investigated and optimized. Absorbance measurements were made at 483 nm. Under the proposed conditions, this method is applicable over concentration range of 4 0 µg ml -1 with molar absorpitivities ranging from.208 x 10 3 1.217 x 10 4 L.mol -1.cm -1 and Sandell s sensitivities ranging from 1.007 x 10-3 2.727 x10-3 µg cm -2. The proposed method was successfully applied for analysis of the cited drugs in formulations and the recovery percentages ranged from 99.47 to 99.8%. The results obtained demonstrated that the proposed method is equally accurate, precise and reproducible as the reported methods thus it is recommended for quality control and routine analysis where time, cost effectiveness and high specificity of analytical techniques are of great importance. Keywords: Spectrophotometry, Triphenyltetrazolium Chloride, Pharmaceutical Analysis, Validation. Introduction Cephalosporins are penicillinase resistant antibiotics derived from moulds of Cephalosporium species. They differ in their antimicrobial spectrum, resistance to β lactamase and method of administration 1. In this work two cephalosporins have been determined; cefepime HCl and cefuroxime Na. Several procedures were reported in literature for their determination e.g. spectrophotometry 2-, fluorometry,7, polarography 8, IR spectroscopy 9, HPLC 10, potentiometry 11 and FIA 12. The present work develops a colorimetric method for the determination of the studied drugs using TTC as an oxidizing agent. The drugs under study were previously determined using ammonium molybdate 13 and 1,10 phenanthroline ferric reagent 14 as oxidizing agents depending on their reducing properties the author s previous work. The purpose of this study was to determine the two drugs in their bulk and dosage forms without prior extraction by simple and selective assays for quality control and routine analysis purposes using TTC as an oxidizing agent. This paper presents a validated
2222 MARWA S. ELAZAZY et al. colorimetric method. The validation was performed in respect of the ICH Q2R1 regulations 1 for analytical procedures. The following parameters were taken into account: specificity, linearity, limits of detection and quantification, accuracy, precision. Experimental A Shimadzu 20 - UV recording spectrophotometer with 10 mm quartz cell was used for all absorbance measurements. Materials and reagents All chemicals were of analytical grade and doubly distilled water was used throughout all measurements.cefepime hydrochloride (Bristol Myers Squibb, Egypt), Cefuroxime sodium (Glaxo Wellcome Egypt), Triphenyltetrazolium chloride (TTC) (Aldrich, Germany), 0.% w/v aqueous solutions, prepared and stored in dark closed bottles, Sodium hydroxide (El-Nasr Chemical Co., Egypt); 0.2 N ethanolic solution. Standard solutions of antibiotics were freshly prepared by dissolving the appropriate amounts of each in methanol to form 1 mg ml -1 solutions. Working solutions of 20 µg ml -1 were also prepared in methanol. Procedure For pure pharmaceuticals Accurately measured aliquots of the standard drug solutions ranging from 40-00 µg ml -1 were transferred into separate 10 ml volumetric flasks, followed by 0.3 ml aliquots of 0.% w/v TTC reagent and 0.3-1 ml of 0.2 N NaOH. The reaction mixture was heated at 7 C in water bath for 10 minutes. Volume was made up to the mark with methanol. The absorbance was measured at the specified wavelength (Table 1) and the concentration was then determined from calibration graphs or the corresponding regression equations. For vials The contents of two vials were mixed well. An amount of powder equivalent to 0 mg of the active ingredient was transferred into 0 ml volumetric flask and the volume was made up to the mark with methanol. Twenty ml of this solution were further diluted to 100 ml with methanol. Drug content in the obtained solution was determined following the general procedures. For tablets Ten tablets were accurately weighed and ground into a fine powder. A weight of powder equivalent to 100 mg of the drug was transferred into 100 ml volumetric flask and extracted with 3x2 ml portions of methanol. These portions were filtered into 100 ml calibrated flasks then the volume was made up to the mark with the suitable solvent. The drug content in the obtained extract was determined following the general procedures. Results and Discussion Tetrazolium salts have been used for colorimetric determination of many compounds of pharmaceutical interest. Examples of these compounds included steroids (based on presence of a side chain with a reducible α-ketol group) 1, carbidopa, methyldopa and ethamsylate (based on presence of two phenyl hydroxy groups) 17-19, the reduction of tetrazolium blue is represented by the following (Scheme 1) 20,21 :
Absorbance Validated Spectrophotometric Assay of Cefepime Hydrochloride 2222 Scheme 1. Reduction of tetrazolium blue to formazan red. The absorption spectra of the reaction product were scanned in the range of 300 0 nm against a reagent blank, characteristic absorption maxima at 483 nm were observed (Figure 1). The rate of reaction is indicated by the rate of color development, which depends on the rate of reduction of TTC by the studied drugs. To optimize conditions, we have investigated a number of parameters such as alkalinity, temperature, time, reagent volume and solvent. Wavelength, nm Figure 1. Absorption spectra of the reaction product resulting from reaction of (1) 0 µg/ml cefepime HCl measured against reagent blank (3) and (2) 20 µg/ml cefuroxime Na measured against reagent blank (4) with TTC. To investigate the effect of alkalinity on the absorbance of reaction product (formazan red), sodium carbonate, sodium bicarbonate, potassium hydroxide and sodium hydroxide were tested. Sodium hydroxide was the best for maximum color development and stability. The optimum basic media was achieved using 0.3 1 ml of 0.2 N NaOH. It was found that the oxidation reduction reaction does not occur in acidic and neutral media. As an assay solvent, methanol afforded maximum sensitivity. For the other solvents tested (ethanol, acetone, propanol, water and water methanol mixture), the color development does not achieve the same wavelength, color intensity and stability. The color obtained was found to be stable for at least 30 minutes at room temperature (Table 1).
2222 MARWA S. ELAZAZY et al. Drug Cefepime HCl Cefuroxime Na Table 1. Analytical data for determination of investigated drugs. max, nm 483 483 Reaction Time, min 10 10 Reaction temperature, C 7 7 Volume of TTC, ml 0.3 0.3 Volume of NaOH, ml 1 0.3 Stability, min 30 0 Identification of validation characteristics Linearity A linear correlation was found between absorbance and concentration in the range given in Table 2. The correlation coefficients, intercepts and slopes for the calibration data for the tested drugs were calculated using least squares method. Table 2. Optical characteristics for the reaction of cited drugs with triphenyltetrazolium chloride (TTC). * Regression equation :- A = a+ bc a = intercept b = slope c = concentration (µg ml -1 ) A = absorbance unit. Parameter Cefepime HCl Cefuroxime Na Beer s law limits, µg ml -1 10-0 4.0-30 Molar absorptivity, L mol -1 cm -1 0.21x10 4 1.217x10 4 Sandell s sensitivity, µg cm -2 1.007x10-3 2.727x10-3 Regression equation (A) Slope, b * 0.010 0.0272 Intercept, a * - 0.0138 2.798x10-4 Correlation coefficient (r) * 0.9999 0.9999 Limit of detection, µg ml -1 1.097 0.280 Limit of quantification, µg ml -1 3. 0.934 Sensitivity The limit of detection (LOD) and limit of quantization (LOQ) for the proposed methods were calculated using the following equations 1 : LOD = 3.3 σ A / B LOQ = 10 σ A / B, where σ A is the standard deviation of replicate determination values under the same conditions as for the sample analysis in the absence of the analyte and B is the slope of the calibration line. In accordance with the formula, the LODs were found to be 0.298 and 0.23 μg ml 1 for cefepime HCl and cefuroxime Na, respectively. The LOQs were 0.903 and 0.79 μg ml 1 for cefepime HCl and cefuroxime Na, respectively. These values indicate high sensitivity of the proposed methods, Table 2. Quantification, accuracy and precision The mean Sandell sensitivity (S) and molar absorpitivity (ε) for each drug were calculated, Table 2. The accuracy was studied using known amounts of the drugs in the range of 4-0 μg/ml, via the recovery coefficient, which was in the range of 99.43-100.42%. The validity of the proposed methods was assessed by applying both the calibration method and the standard addition technique, Tables 3,4.
Validated Spectrophotometric Assay of Cefepime Hydrochloride 2222 Table 3. Comparative analytical results of the proposed and the reported methods for the tested drugs in pure forms: Statistical parameters ** Cefepime Hydrochloride Cefuroxime Sodium Proposed method Reference method 14 Proposed method Reference method 13 *Mean% Recovery * n * Variance * S.D. *S.E. *R.S.D. * t * f 100.01 7 0.17 0.409 0.1 0.409 1.0(2.228) 2.83 (4.3) 99.81 0.09 0.243 0.109 0.243 100.12 8 0. 0.74 0.23 0.744 0.447(2.201) 3.8 (4.12) 99.98 0.144 0.380 0.170 0.380 ** n = Number of experiments S.D. = Standard Deviation S.E. = Standard Error R.S.D.= Relative Standard Deviation t = t test of unpaired data F = Variance test. Table 4. Comparative analytical results of the proposed and the reported methods for tested drug in some pharmaceutical formulations: Formulation Maxipime vials (00 mg cefepime HCl/ vial, Bristol Myers Squibb, Egypt) Zinnat tablets (20 mg cefuroxime Na/ tablet, Glaxo Wellcome Egypt) Zinnat vials (20 mg cefuroxime Na/ vial, Glaxo Wellcome Egypt) Statistical parameters ** Mean % recovery n Variance S.D. S.E. R.S.D. t F Mean %recovery n Variance S.D. S.E. R.S.D. t F Mean % recovery n Variance S.D. S.E. R.S.D. t F Proposed method Calibration Standard method addition method 100.33 100.31 7 0.24 0.02 0.14 0.77 0.210 0.293 0.12 0.774 1.94 (2.228) 1.497 (2.228) 1.74 (4.3) 3.9 (4.3) 99.43 0.43 0.737 0.301 0.741 0.097 (2.22) 1.38 (.19) 99.9 0.18 0.431 0.17 0.431 0.17 (2.22) 1.430 (.19) 99.73 1.309 1.144 0.47 1.147 0.477 (2.22) 0.912 (.19) 100.42 0.23 0.48 0.198 0.484 2.038 (2.22) 1.127 (.19) Reference method 99.8 14 0.12 0.390 0.174 0.391 99.47 13 0.397 0.30 0.282 0.33 99.8 13 0.2 0.1 0.231 0.17 ** n = Number of experiments S.D. = Standard Deviation S.E. = Standard Error R.S.D.= Relative Standard Deviation t = t test of unpaired data F = Variance test.
2222 MARWA S. ELAZAZY et al. Reproducibility The reproducibility of the proposed methods was determined by replicate analysis of five separate solutions of the working standards. The methods gave satisfactory results; the relative standard deviations were 4.33 and 3.83% for cefepime HCl and cefuroxime Na, respectively, Table, indicating good reproducibility of the proposed methods. This precision level is adequate for the precision and routine analysis of the cited drugs in quality control laboratories. Table. Replicate analysis of the examined drug solutions by the proposed method. Absorbance Sample Number Cefepime HCl, Cefuroxime Na, 0 μg ml 1 20 μg ml 1 1 0.210 0.83 2 0.211 0.43 3 0.198 0.7 4 0.200 0.0 0.220 0.94 Mean 0.208 0.9 SD 0.009 0.0218 RSD 4.33 3.83 ** S.D. = Standard Deviation R.S.D.= Relative Standard Deviation. Robustness Robustness was assessed as the method was developed. Reliable results can be obtained with small variations in the methodology by evaluating the influence of small change in the experimental parameters on the analytical performance of the proposed methods 22. As a general rule, one parameter was changed whereas the others were kept unchanged, and the recovery percentage was calculated each time. Variations in the quantities of reagents added over the range % had no effect on optical density, which indicates the robustness of the proposed methods. Conclusion The present method besides its sensitivity, selectivity and simplicity, offers many advantages such as: a. No prior extraction or separation of the studied drugs is necessary, b. The precision, accuracy and reproducibility of the results are comparable to those of the official and reference methods These advantages render the proposed method eminently suited to routine determination of the studied drugs in various pharmaceutical formulations with low cost depending on availability of chemicals and equipments. References 1. Martindale, The Complete Drug Reference, 32 nd Ed., 1999. 2. Morelli B, J Pharm Biomed Anal., 1988,, 199-209. 3. Morelli B, Anal., 1988, 113, 1077-1082. 4. Rodenas V, Parra A, Garcia Villanova J and Gomez M D, J Pharm Biomed Anal., 199, 13 (9), 109 1099.. Amin A S, Khalil H M and Saleh H M, Sci Pharm., 2001, 9, 143-10.
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