Spectrophotometric Estimation of Cefuroxime and Ceftazidime in Bulk and in Dosage Forms

Three simple, accurate and sensitive spectrophotometric methods (A, B and C) for the determination of cefuroxime and ceftazidime in bulk samples and in dosage forms are described. They are based on the reaction with nitrous acid forming a nitroso derivatives which can be measured at A,, 350 and 355 nrn for cefuroxime (I) and ceftazidime (11), respectively (method A) or by oxidation of drug I or I1 with an excess of freshly prepared hypobromite and the residual hypobromite was treated with sodium fluorescein at the optimum experimental conditions and measured at A,,,, at 5 17 nm (method B). Method C is based on the formation of tris (0-phenanthroline) iron(I1) complex (ferroin) upon the oxidation of the studied drug I or I1 with an iron (111)-ophenanthroline mixture in acetate buffer solution of pH 3.6 and measuring at h,, 509 nm. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges 0.2 6.0,0.2 3.2 and 0.1 5.6 pg ml-' for methods A, B and C, respectively. The apparent molar absorptivity, Sandell sensitivity, detection and quantitation limits were calculated. For more accurate results, Ringbom optimum concentration range was 0.2 5.6 pg ml-I. The validity of the proposed methods was tested by analysing dosage forms containing the studied drugs I and 11. The relative standard deviations were i 1.25% with recoveries 98.6 101.4% . Key wor-ds: Cephalosporins determination, spectrophotometry, ferroin complex, nitroso derivatives, hypobromite. Introduction Cephalosporins are a large class of antibiotic agents which are derived from microorganism cephalosriutn acremonium. They resemble penicillin in structure with variation in chemical structure next to beta lactam ring. Despite this similarity to penicillin, they offer broader anti~l~icrobial coverage. A.S. A m i n , H.M. K h a I I i I and H.M. S a I e h : Several procedures have been reported in the literature for the analysis of cephalosporins. These methods are spectrophotometry('"), polarography(7'8), capillary zone electrophoresis"', irnrnunoassay analysis('9 and high performance liquid chromatography (HPLC) ('I9 21) In the present work, three simple, accurate and sensitive methods for the determination of cefuroxime and ceftazidime in the presence of various excipients and diluents are developed. These methods are also applicable for determination of the two mentioned drugs in some pharmaceutical dosage forms without previous separation. Experimental Apparutus A Shimadzu 260 UV-Visible spectrophotometer with 10 mm matched quartz cells was used for all the absorbance measurements. An Orion research model 601Aldigital ionalyzer pH-meter was used for check the pH of all solutions. Reagents All chemicals were of analytical or pharmacopoeia1 grade and double distilled water was used throughout. Aqueous solutions of sodium nitrite (20%, WN), sodium hydroxide (20%, W/V), hydrochloric acid (5 and lo%, V N ) , and ammonium chloride (1 5%,WN) were prepared for method A. Aqueous solution of potassium hypobromite was prepared by addition of 12 ml 20% HCl to 6.0 ml of 0.1 M potassium bromate in 5% potassium bromide, then sufficient amount of 0.5M NaOH was added to produce hypobromite and complete the volume to 250 ml with bidistilled water in a 250 ml calibrated flask. Sodium fluorescein was prepared by dissolving 0.3803 g sodium fluorescein (BDH, Pool, UK) in water and completed to the mark in a one liter calibrated fask with the same solvent. Acetate buffer solutions of pH ranges 2.16-5.85 were prepared as recommended previously(22) [for Method B] . 1,lO-Phenanthroline-iron(II1) colour reagent was freshly prepared by mixing 0.198 g of 1,lO-phenanthroline (Sigma Chemicals, St. Louis, USA), 2.0 ml of 1.0 mol1L hydrochloric acid and 0.160 g of ferric ammonium sulphate (BDH, Pool, UK) and diluting with water to 100 ml in a 100 ml calibrated flask. This reagent is stable for more than 6.0 weeks if stored in a dark bottle a way from light (for method C). Prepuralion qf slandurd stock drug solution. Standard cefuroxim sodium and cefazidime (Glaxo, Egypt, SAE, Cairo, Under the licence from Glaxo group, England) [lmg I ml] was prepared by dissolving 100 mg in Spectrophotometric Estimation of Cefuroxime and Ceftazidime in Bulk ... 145 100 ml distilled water. The working standard solutions were obtained by further dilution of stock solution with water. Analysis ofpure sample Method A Aliquots of standard drug solution equivalent to 2.0-60 pg were pipetted into a series of 1


Introduction
Cephalosporins are a large class of antibiotic agents which are derived from microorganism cephalosriutn acremonium.They resemble penicillin in structure with variation in chemical structure next to beta lactam ring.Despite this similarity to penicillin, they offer broader anti~l~icrobial coverage.
Several procedures have been reported in the literature for the analysis of cephalosporins.These methods are spectrophotometry('"), polarography(7'8), capillary zone electrophoresis"', irnrnunoassay analysis('9 and high performance liquid chromatography (HPLC) ('I9 21)   In the present work, three simple, accurate and sensitive methods for the determination of cefuroxime and ceftazidime in the presence of various excipients and diluents are developed.These methods are also applicable for determination of the two mentioned drugs in some pharmaceutical dosage forms without previous separation.

Apparutus
A Shimadzu 260 UV-Visible spectrophotometer with 10 mm matched quartz cells was used for all the absorbance measurements.An Orion research model 601Aldigital ionalyzer pH-meter was used for check the pH of all solutions.

Reagents
All chemicals were of analytical or pharmacopoeia1 grade and double distilled water was used throughout.
Aqueous solution of potassium hypobromite was prepared by addition of 12 ml 20% HCl to 6.0 ml of 0.1 M potassium bromate in 5% potassium bromide, then sufficient amount of 0.5M NaOH was added to produce hypobromite and complete the volume to 250 ml with bidistilled water in a 250 ml calibrated flask.Sodium fluorescein was prepared by dissolving 0.3803 g sodium fluorescein (BDH, Pool, UK) in water and completed to the mark in a one liter calibrated fask with the same solvent.Acetate buffer solutions of pH ranges 2.16-5.85were prepared as recommended previously (22) [for Method B] .
1,lO-Phenanthroline-iron(II1) colour reagent was freshly prepared by mixing 0.198 g of 1,lO-phenanthroline (Sigma Chemicals, St. Louis, USA), 2.0 ml of 1.0 mol1L hydrochloric acid and 0.160 g of ferric ammonium sulphate (BDH, Pool, UK) and diluting with water to 100 ml in a 100 ml calibrated flask.This reagent is stable for more than 6.0 weeks if stored in a dark bottle a way from light (for method C).
Standard cefuroxim sodium and cefazidime (Glaxo, Egypt, SAE, Cairo, Under the licence from Glaxo group, England) [lmg I ml] was prepared by dissolving 100 mg in 100 ml distilled water.The working standard solutions were obtained by further dilution of stock solution with water.

Analysis ofpure sample Method A
Aliquots of standard drug solution equivalent to 2.0-60 pg were pipetted into a series of 10 1111 calibrated flasks.1.0 ml of 5.0 % HCl followed by 1.0 ml of 20% Sodium nitrite were added, and heated in a water bath at 60 O C for 10 min.The mixture was cooled, then a dropwise of 5.0 ml of 15% NH4C1 was added under stirring condition.2.0 ml of 20 % NaOH was added and the volume was completed to the mark with water.The absorbance was measured at 350 and 355 nm for drug I and 11, respectively, against a reagent blank prepared similarly without drug.
For ceftazidime the yellow colour developed spontaneously on cold, whereas heating for 10 min in waterbath at 60 O C is necessary for production of yellow colour for cefuroxime.

Method B
Aliquots of standard solutions containing drugs I and I1 in the ranges 5.0-80 pg were transferred into 25 ml calibrated flasks, 2.0 ml of potassium hypobromite reagent was added.After five mins, 2.0 ml of acetate buffer solutions of pH 5.5 and 5.7 for I and 11, respectively, were added followed by 1.0 ml sodium fluorescein reagent.The volume was completed to the mark with water and the absorbance was measured at 5 17 nrn after 10 min against a reagent blank prepared similarlly.

Method C
Aliquots of the sample solutions containing 0.1-56 pg were pipetted and transferred into a series of 10 ml calibrated flasks.2.0 ml of 1 ,lo-phenanthrolineiron(II1) reagent was added and the mixture was heated at 75 "C for 5.0 min and cooled.The solution was completed to the mark with water and the absorbance was measured at A , , , 509 nm against a reagent blank prepared in the same way without drug.

Analysis of phamaceutical dosage forms (Vials)
The content of 10 vials was mixed and weighed.The average weigh of one vail was calculated.20 mg of the mixed powder was dissolved in 10 ml water and then completed to the mark with water in 50 ml calibrated flask.This solution was used after appropriate dilution with water and continued as mentioned above for methods A, B and C.

Results and discussion
The absorption spectra of the reaction products in methods A, B and C show characteristic h,,,,, values (Fig. 1).The experimental reaction conditions were stablished by variation of one parameter at a time(*".

hbs.
Fig ( 1) Absorbance spectra for method A (AI and All), method B (BI and BIr) And method C (CI and CII) using drug I and 11, respectivily In method A, 0.8-1.2ml of each of 5.0% HCl and NaN02 were the optimum concentration ranges for complete nitrosation on cold for ceftazidime and after heating in a water bath at 60°C for 7.0-15 min for cafuroxime.Hence, 1.0 ml of both 5.0% HC1 and NaN02 was added and heated for 10 min at 60 OC were chosen for drug I.For maximum absorbance 4.0-7.0ml of 15% NH4Cl and 1.5-2.4ml of 20% NaOH was necessary for completation of nitrosation reaction.5.0 ml of 15% NH4Cl and 2.0 ml of 20% NaOH were selected for the procedure.Maximum colour intensity was attained 5.0 min after the addition of NaOH and the coloured species was stable for 4.0 h.Method B involves two stages: oxidation of drug I or I1 with excess of freshly prepared hypobromite and the residual hypobro~nite was treated with sodium fluorescein in buffered medium of pH 5.5 as optimal, where equivalent eosin was produced with A , , , at 5 17 nm.2.0 ml of both potassium hypobromite and of acetate buffere of pH 5.5 and 5.7 for drug I and 11, respecti1,ely was the optimum for the first stage.For the second one, 1.0 ml of sodium fluorescein reagent was found optimal, since the results were highly concordant at these conditions.In Method C, 1.6-2.5 ml of 1,lOphenanthroline-iron(II1) reagent with heating at 75 "C for 10 min, was found to give maximum colour intensity.The order of addition was found not to have a significant effect.The coloured species (ferroin) was stable for more than 12 h.

Analytical duta
Beer's law limits, molar absorptivities, regression equations and correlationcoefficients obtained by linear least squares treatment of the results are given in Table 1.The precision and accuracy were found by analysis of six separate samples containing known amounts of the drug (4.0 pg for method A, 2.0 pg for method B and 3.0 pg for method C) and the results are summarized in the same Table .The relative standard deviation 1.25% and +1.4% range of error at the 95% confidence level are also given.
The values obtained by the proposed and reference ("'24) methods for pure drugs I and I1 are compared in view of accuracy (t-test) and precision (F-value)"').The results are in good agreement with lower values of t-and F-tests (Table 1) compared with the theoretical values indicating that there is no significant difference between the proposed and reference methods (21"1).

3.09
Thc detection and quantification limits were calculated from the standard deviation of the absorbance measurements obtained from series of 13 blank solutions for each procedures.The limits of detection (K=3) and of quantification (K= 10) were established according to IUPAC definitions(*".

Effecf oj'inferjiring species
The criterion of interference was an error of more than 3.0 % in absorbance.Experiments showed that there was no interference from additives and excipients e.g.lactose, glucose, fractose, calcium hydrogen phosphate, magnesium stearate and starch for the examined methods A, B and C. Also, there was no interference from cornillon degradation products resulted from thermal decon~position of drug I and I1 indicating the ability to use the proposed methods in routine quality control for phamaceutical dosage forms.

Analytical applications
The proposed methods were applied to determine drug I and I1 in pharmaceutical dosage forms (commercial products randomly collected from local pharmacies).Table 2 list the results obtained by the proposed and reference methods (2 1,21) [based on HPLC and A 1 % in case of cefuroxime and ceftazidime, respectively].The results indicate good agreement with the reference method.The proposed methods can be recommended for routine analysis in the majority of drug quality control laboratories.Another favourable characteristic of the methods is the stability of absorbance of the coloured species formed which are stable for 4.0,6.0 and 12 h, using methods A, B and C, respectively.
On compariny the obtained results by the proposed methods with those of the reference methods(21* 'I, the t-test for accuracy and F-values for precision assessment(25' was used.The calculated values did not exceed the corresponding theoretical values, indicating insignificant difference between results.The proposed methods were more accurate, with high recoveries amounting to 99.8+1.2%,100.3+_1.3% and 100.5+_1 .l%for method A, B and C, respectively, compared with 100.8f 1.7% using the reference methods (21* 24).

Table 2 :
statistical analysis of the results obtained by applying the proposed methods to pharmaceutical preparations.a-value in paranthesis are the theoretical value of t and F-value for Five degree of freedom and 95% confidence limit.