Colorimetric and Titrimetric Methods for Determination of Some Cephalosporins in their Pure and Dosage Forms

Two colorimetric and titrimetric procedures are developed for determination of some cephalosporins, namely, cefotaxime sodium (CFT), cehroxime sodium (CFU) and ceftazidime pentahydrate (CFZ). The first method, a colorimetric method, was based on the reaction of these cited drugs with 4-chloro-7nitrobenzofurazan (NBD-Cl), in the presence of 5 x lo-' M borax, with the formation of stable colored chromogens with maximum absorbance in the range of 409 414 nm. All variables affecting the development of the colour have been investigated and the conditions were optimized. The method obeys Beer's law over concentration ranges of 19.09 95.49, 17.85 124.98 and 63.66 254.66 pg ml" of CFT, CFU and CFZ, respectively. Obedience to Beer's law permitted the successful application of the proposed method for the assay of cefuran, cefumax and fortum vials. The results obtained reveal good percentage recoveries, which are in good agreement with those obtained by the official methods. In the second method, the titrimetric method, the conditions for the oxidimetric titration of cephalosporins have been studied. The titration takes place because of catalytic quantities of KBr, which give rise to an intermediate that can be hydrolyzed to the sulfoxide. The operating conditions are described and a hypothesis is put forward concerning the mechanism of the reaction.

NBD-Cl has been reported as fluoregenic reagent for the determination of amines (13).Thiocompounds have been reported to form intensely coloured products in an alkaline medium with NBD-Cl which could be used for their colorimetric determination (14).Therefore, it was decided, on the basis of these facts, to develop a quantitative method for determination of the previous mentioned cephalosporins with NBD-Cl, which may be used for their analysis either in pure form or in pharmaceutical formulations.
On the other hand, with the aim of investigating the nucleophilic character of thioethers of pharmaceutical interest, and in order to develop rapid and precise methods for the analysis of products and intermediates containing the R-S-RI group, we study the possibility of titrating cephalosporins by potentiometric techniques.The ability of thioethers to take part in addition reactions with halogens, forming derivatives of the type: is well known.In an aqueous acidic environment these derivatives give rise to the corresponding sulfoxides.Bromine (or NBS) readily forms compounds of the type indicated above (better than the other halogens) under ordinary reaction conditions.If the hypothesis concerning the possible formation of dihalogen derivatives were valid for all sulfides, then even the most unreactive thioethers should be readily oxidized to sulfoxides with an oxidizing agent of appropriate strength, such as a salt of tetravalent lead for example, provided that traces of bromine were present (1 5).Rather than using bromine itself, we should also obtain the same results with KBr, which give rise to bromine in an oxidizing medium.
The propose of the present work is to describe the development of two simple and accurate colorimetric and titrimetric methods for the analysis of three cephalosporin derivatives.

111-Standard solutions:
1. Solutions for colorimetric determination: 2 x 10" M solution of CFT, CFU and CFZ was prepared by dissolving 95.49, 89.27 and 127.33 mg, respectively in 10 ml bidistilled water, mixing with 10 ml 0.1 N NaOH and placing in water bath at 95 O C for 25 min.After hydrolysis, the mixture is cooled, neutralized to pH 7 with 0.1 N HC1 and diluted to 100 ml with bidistilled water.

Solutions for titrimetric determination:
1.75 x M solution of CFT, CFU and CFZ was prepared by dissolving 83.55, 78.8 1 and 1 1 1.40 mg, respectively in 10 ml bidistilled water.

PROCEDURES A-Colorimetric procedure:
Different aliquots of the standard drug solution in the concentration range stated in table 1, were placed in 10-ml volumetric flasks, followed by 2 ml of 5 x lo-' M borax and 0.1 ml of 2 x 10" M of NBD-Cl, mixed well and allowed to stand for 30 min.at room temperature.1 ml 0.1 N HC1 was added and the volume was completed with methanol.The absorbance was measured at 409 -414 nm against blank in which the drug was omitted.

A-Colorimetric method
In this work, the reaction of the hydrolysis product of the three cephalosporins, which have free -SH group (Scheme I), with NBD-C1 reagent was studied.

Scheme 1
The reaction of cephalosporins with NBD-CI.

I t
The method is based on the high nucleophilicity of thiol group and the ability of NBD-CI to couple with it through SN2 reaction giving yellow colour with A,,,, at 414 nm (Figure 1).Wavelength (nm)

Figure (1)
Absorption spectra of 53.56 pgml" cefuroxime sodium (. . . . . . . . ..), 0.1 ml of 0.02 M NBD-C1 (----) and their reaction product in methanol ( ) All conditions studied were optimized at room temperature (25 k 5'C).A volume of 0.04, 0.04 or 0.1 ml of NBD-Cl solution (2x10'~ M) in acetone was found to be satisfactory for CFT, CFZ and CFU, respectively.Large excess of reagent was followed by disappearance of the peak at the specific A , , and the appearance of another peak at 378 nm.Owing to the presence of labile chloride, a daily fresh solution is prepared.To generate nucleophile(s) from the studied drugs, 2 ml of 5x10" M of borax was used.The time required for the reaction to be completed was found to be 30 min., and the colour was stable for 24 h.Addition of 1 ml of 0.1 N HCl was essential to stabilize the chromogen formed and neutralize the excess borax.Cifferent dilution solvents have been tried (Table 2), methanol, isopropanol and acetonitrile gave best results.Concerning the stoichiometric relationships, the molar ratio method indicated a molar ratio 2 : 1 cephalosporins (thiol) to NBD-CI.The method described above was applied to the cyclic thioether contained in the cephalosporins.Figure 2 show the potentiometric titration curve of cefuroxime sodium with 0.1 N Pb(CH3C00)4 in 70 % acetic acid in absence and presence of KBr (mole ratio of KBr to cephalosporins of 1 : 1, 1 :25, 1 :50 and 1 : 100).As to the titration, the same results is obtained with bromine water, NBS, or potassium bromide.For practical reasons we used potassium bromide.For a good titration the minimum quantity of the catalyst can be used is 1 mol of potassium bromide to 1 mol of cephalosporins.The explanation for this reaction may be as follows:

+ S=O + 2 HBr
The hydrobromic acid liberated is continuously oxidized by the oxidizing agent added during the titration and is immediately used again.Because of the titration curve does not permit a clear differentiation between the potential jump associated with oxidation of cephalosporins and that associated with oxidation of the bromide, it is acceptable, for analytical purposes, to carry out a blank titration with the solvent containing the appropriate amount of catalyst and to subtract the corresponding volume of titrant from the subsequent titration.

Ouantification, accuracy and precision:
For the colorimetric method a linear correlation was found between absorbance and concentration in the ranges given in table 1.The correlation coefficients, intercepts and slopes for the calibration data for the three cited drugs are calculated using the least-squares method.The precision and accuracy of the two methods were tested by estimating five replicates of the three cephalosporins.The percentage standard deviation and the standard analytical error, can be considered to be very satisfactory (Table 3 & 4).
The utility of each method was verified by means of replicate measurements of the pharmaceutical formulations and recovery experiments.Recoveries were determined by adding standard drug to the pre-analyzed mixture of pharmaceutical preparations.The results of recovery experiments by the proposed methods are listed in Table 4.The performance of the methods was assessed by caIculation of the t-and Fvalues compared with the official method (12) and for The results showed that the calculated t-and Fvalues did not exceed the theoretical values (95% confidence limits for five degree of freedom), Table 4 from which we can conclude that the proposed methods do not differ significantly from official method (1 2).

Conclusion:
The data given above reveal that the proposed methods are simple, accurate and sensitive with good precision and accuracy.With these methods, one can do the analysis with speed at law cost without losing accuracy.The proposed methods can be used as alternative methods to reported ones for the routine determination of cefotaxime sodium, cefuroxime sodium and ceftazidime pentahydrate in the pure form and in pharmaceutical vials.

Table ( 1 ):
Parameters for the spectrophotometric determination of cefotaxime sodium, cefuroxime sodium and ceftazidime pentahydrate using NBD-CI B-Titrimetric procedure: Parameters Beer's law limits (pgml-l) Molar absorptivity (mol-'cm-') Regression equation: Slope (b) Intercept (a) Correlation coefficient (r) Relative standard deviation (%) 1 ml of standard solution for titrimetric procedure was dissolved in 70 ml of a 70 % solution of acetic acid water.2.5 x M of KBr was added and the solution was titrated with 0.1 N lead tetraacetate solution.The titrant was added at a rate of 1 ml/ A = a + b C a = intercept b = slope C = concentration in pgml-'

A-Shimadzu 260 UV recording spectrophotometer B-Jenway 30 1 0 pH meter C-Double junction platinum electrode 11-Materials and reagents
and the end point was detected potentioinetrically.The reaction rate was comparable to that of the usual neutralization reactions min

Table ( 2
) -Effect of solvent on the absorption intensity of reaction product of cefuroxime sodium

Table ( 3
): Evalution of the accuracy and precision of the titrimetric procedure * Mean + standard deviation for five determinations

Table ( 4
(12)etermination of cefotaxime sodium, cefuroxime sodium and cefiazidime pentahydrate in commercial preparation using the proposed methods compared statistically with an official methods(12) a USP SX111 ( 12) b .