Spectophotometric method for determination of certain cephalosporins using 4-chloro-7-nitrobenzo-2-oxa-1 , 3-diazole ( NBD-Cl )

A simple, accurate and precise spectrophotometric method has been proposed for the determination of eleven cephalosporins, namely; cefaclor monohydrate, cefadroxil monohydrate, cefalexin anhydrous, cefradine anhydrous, cefotaxime sodium, cefoperazone sodium, ceftriaxone sodium, ceftazidime penthydrate, cefazolin sodium, cefixime and cefpodoxime proxetil in bulk drug and in pharmaceutical formulations. The method depends on hydrolysis of the studied drugs using 0.5M NaOH at 100°C and subsequent reaction of the formed sulfide ions with NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole) to form a yellow-colored chromogen measured at 390 nm. Different variables affecting the reaction (e.g. NaOH concentration, hydrolysis time, NBD-Cl concentration and diluting solvent) were studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.99900.9999) were found in the range of 5-160 μg mL for all studied drugs. The limits of assay detection and quantitiation ranged from 0.289 to 5.867 and from 0.878 to 17.778 μg mL; respectively. The accuracy and precision of the proposed method were satisfactory. The method was successfully applied for analysis of the studied drugs in their pharmaceutical formulations and the recovery percentages ranged from 96.6 to 103.5%.

The hydrolytic degradation of cephalosporins was very often used as a preliminary step in the analytical procedure used for their determinations [25][26][27][28][29][30][31][32].The literature reveals that many spectrophotometric methods were developed for cephalosporins determinations that based on hydrolysis of these drugs using alkaline degra-   dation and subsequent reaction of the formed sulfide ions with chromogenic reagents [26,27].NBD-Cl (Figure 2) has been reported as fluorogenic reagent for determination of amines [33] and for spectrophotometric determination of many compounds [34][35][36][37][38][39][40][41].Thiocompounds have been reported to form intensely colored products in an alkaline medium with NBD-Cl which could be used for their colorimetric determination [42].It is always required to develop analytical methods using low cost techniques.UV-Vis spectrophotometry is still considered a convenient and economical technique for routine analysis of drugs in pharmaceutical formulations.On the basis of the aforementioned reasons, it was decided to develop a quantitative method for the determination of the studied cephalosporins based on their alkaline hydrolysis and subsequent reaction of the resulting hydrolysates with NBD-Cl, which may be used for their analysis either in pure forms or in pharmaceutical formulations.This method is selective for cephalosporins, since other β-lactam antibiotics such as penicillins do not give sulfide ions under the degradation conditions employed [27,[43][44][45].

Preparation of Standard Solutions
Stock solutions containing 100 mg mL -1 of each cephalosporin were prepared in double distilled water (methanol was used in case of cefadroxil monohydrate, cefalexin anhydrous, cefaclor monohydrate, cefradine anhydrous, cepodoxime proxetil and cefixime).Working standard solutions containing 0.5-2.5 mg mL -1 (in case of cefadroxil monohydrate and cefalexin anhydrous), 1-6 mg mL -1 (in case of cefradine anhydrous), 2-8 mg mL -1 (in case of cefaclor monohydrate, cefazolin sodium, cefotaxime sodium, ceftriaxone sodium and cefpodoxime proxetil), 2-10 mg mL -1 (in case of cefixime) and 2-16 mg mL -1 (in case of cefoperazone sodium and ceftazidime pentahydrate) were prepared by suitable dilution of the stock solution with double distilled water (in case of cefadroxil monohydrate, cefalexin anhydrous, cefaclor monohydrate, cepodoxime proxetil and cefixime, dilution was made using methanol).The stock and working standard solutions must be freshly prepared.

Preparation of Sample Solutions
Tablets and capsules.Twenty tablets or the contents of 20 capsules were weighed, finely powdered and mixed thoroughly.An accurately weighed amount of the powder obtained from tablets or capsules equivalent to 250 mg of each drug was transferred into a 25-mL volumetric flask, dissolved in about 10 mL double distilled water (10 mL methanol was used in case of cefadroxil monohydrate, cefalexin anhydrous, cefaclor monohydrate, cefradine anhydrous, cepodoxime proxetil and cefixime), sonicated for 15 min, diluted to the mark with double distilled water (in case of cefadroxil monohydrate, cefalexin anhydrous, cefaclor monohydrate, cefradine anhydrous, cefpodoxime proxetil and cefixime, dilution was made using methanol), mixed well and filtered; the first portion of the filtrate was rejected.Further dilutions with the same solvent were made to obtain sample solution containing the specified concentration for each drug as mentioned under the preparation of standard solutions and then the general procedure was followed.
Vials and powder for oral suspension.An accurately weighed amount of powder equivalent to 250 mg of each drug was transferred into a 25-mL volumetric flask, then the procedure was followed as under tablets and capsules beginning from (dissolved in about 10 mL double distilled water……….).

General Procedure
Accurately measured one milliliter aliquot volume of the standard or sample solutions was transferred into 10-mL volumetric flask.5 mL of 0.5 M NaOH were added and the flask was heated in a boiling water-bath for 30 min, cooled to room temperature and completed to volume with double distilled water.One milliliter of the resulting drug hydrolysate was pipetted into 10-mL volumetric flask, 1.0 mL of 3 × 10 -3 M NBD-Cl was added followed by 1 mL of concentrated HCl.The resulting solution was mixed well and the flask was completed to volume with ethanol.The absorbance was measured at 390 nm against reagent blank treated similarly.

Absorption Spectra
As shown in Figure 3, the absorption spectrum of NBD-Cl in acetone shows a maximum absorption at 340 nm.All the investigated drugs after alkaline hydrolysis give a very weak absorption taking cefalexin anhydrous hydrolysate as a representative example which gives a very broad absorption maximum at 350 nm.The interaction colored product of cefalexin anhydrous hydrolysate with NBD-Cl shows absorption maximum at 390 nm (Figure 3).

Optimization of Reaction Variables
Since the developed method depends on the formation of colored product by the interaction of NBD-Cl with sulfide ions resulted from the alkaline degradation of cephalosporins so, optimization studies were carried out extensively to find the optimum conditions for the alkaline degradation and subsequently the optimum yield of sulfide ions and the maximum stability of the chromogen formed taking cefalexin anhydrous (15 μg mL -1 ) as a representative example for these studies.These variables include: Effect of NaOH concentration.The influence of sodium hydroxide concentration on producing the maximum absorption intensity was investigated using 0.1-1.0M NaOH keeping other factors constant.Maximum absorption readings were obtained upon using 0.5 M NaOH; above this concentration and up to 1 M NaOH, the absorbance remains constant.So, this concentration was selected for further work (Figure 4).
Effect of hydrolysis time.The effect of hydrolysis time on the absorption intensity was studied using different heating times in a boiling water bath (at 100°C) starting from 10 min until 2 hours and the reaction was carried out as usual.The obtained absorbance readings were plotted against hydrolysis time.The maximum absorption intensity was attained after 20 min and remained stable for at least 100 min.Thirty minutes hydrolysis time was used in all subsequent experiments as shown in Figure 5.
Effect of NBD-Cl concentration.The concentration of NBD-Cl, for the maximum color development was varied in the range of 0.75 × 10 -3 -4 × 10 -3 M. It was found that 1 mL of 3 × 10 -3 M NBD-Cl was the most suitable con-  centration for determination of the studied drugs as shown in Figure 6.Owing to the presence of labile chloride, a daily fresh solution is recommended.
Effect of type and concentration of acid.Different acids such as sulfuric, hydrochloric, perchloric, nitric and acetic acids were tested to determine the most suitable acid for the reaction.One milliliter of concentrated hydrochloric acid was selected in this study as it gave the highest absorbance readings taking cefalexin anhydrous (15 μg mL -1 ) as a representative example (Table 2).
Further investigations were carried out in order to find the most suitable concentration of hydrochloric acid.It was observed that higher absorbance readings and more reproducible results were obtained upon increasing hydrochloric acid concentration.As a result of these investigations, 1 mL of concentrated hydrochloric acid was used for subsequent work.
Effect of reaction time.The reaction between the investigated drugs hydrolysates and NBD-Cl was very rapid and the interaction colored product can survive before dilution unchanged for at least 1 hour.However, measurements were achieved instantaneously.
Effect of diluting solvent.Different solvents were tested in order to select the most appropriate solvent for optimum color development.The results given in Table 3 show small shifts in the position of the maximum absorption peak.The absorption intensities were slightly influenced.Ethanol was used throughout this work because it gave the highest absorbance readings and the most reproducible results.
Stability of the reaction colored product.Stability time was obtained by following the absorbance readings of the developed reaction product for 24 hours at room temperature (25 ± 5°C).It was found that the produced color was stable for 24 hours for all studied drugs.

Calibration Curves
Linear relationship was obtained for all studied drugs by applying the developed method (Table 4).Good linearity of the calibration curves were clearly evident by excellent correlation coefficients which ranged from 0.9990 to 0.9999 and coefficients of determination ranged from 0.9978 to 0.9998.This wide variation in the linearity range may be attributed to the different yields  of sulfide ions from the studied cephalosporins [45].

Method Validation Study
The method was validated according ICH guidelines on the validation of analytical methods [46] and complied with USP 31 validation guidelines [3].All results were expressed as percentages, where n represents the number of values.For the statistical analysis Excel 2003 (Microsoft Office) was used.A 5% significance level was selected.
LOD and LOQ.The limits of detection and quantitation for all studied drugs ranged from 0.29 to 5.87 and from 0.88 to 17.78 μg mL -1 ; respectively which indicate high sensitivity of the proposed method (Table 4).
Accuracy.The accuracy of the method was determined by investigating the recovery of each of the studied drugs at three concentration levels covering the specified range (six replicates of each concentration).The results shown in Table 5 depict good accuracy and recovery percentage ranged from 98.0 to 102.3%.
Precision.As shown in Table 6, the small values of SD and % RSD point to high precision of the proposed method.
Selectivity.The effect of the presence of common excipients such as; starch, talc, lactose, glucose, sucrose, magnesium-stearate and gum acacia was studied.It was found that no interference was introduced by any of them.
Robustness.Robustness was examined by evaluating the influence of small variation in the experimental parameters on the analytical performance of the proposed method [47].The studied parameters were: NaOH con-centration, NBD-Cl concentration, heating temperature and heating time on the method suitability and sensitivity.It was found that none of these variables significantly affects the performance of the method (Table 7) which indicates the robustness of the proposed method.

Applications to the Analysis of Pharmaceutical Dosage Forms
The proposed method was applied successfully for determination of the studied drugs in their pharmaceutical dosage forms.Six replicate measurements were made in each case, the results obtained were validated by comparison with a previously reported method [48].No significant difference was found by applying t-and F-tests at 95% confidence level indicating good accuracy and precision (Table 8).Recovery studies were also carried out by standard addition method [49].The results in Table 9 indicate good recoveries (96.0 to 103.8%) and confirm that there is no interference from frequently encountered excipients or additives.
In the proposed method, sulfide ions were allowed to react with NBD-Cl via SN 2 mechanism.The high nucleophilicity of sulfide ions, the presence of Cl -anion as       a good leaving group at position 4 in addition to the presence of nitro group as an electron withdrawing group at position 7 of the aromatic ring in NBD-Cl result in replacement of Cl -anion with the attacking sulfide ions which in turn lead to the formation of a yellowcolored chromophore (λ max at 390 nm).The reaction product is stable in strong acidic medium, moreover acidification could minimize possible competition between the generated sulfide nucloephile and excess OH - which may lead to decrease in the chromogen formed.The proposed reaction mechanism is given in the following scheme: The production of sulfide ions was confirmed by carrying out specific qualitative tests such as dilute hydrochloric acid, cadmium acetate, sodium nitroprusside and methyelene blue tests [56] or by comparing λ max of the formed chromogen with that obtained after applying the developed method to sodium sulfide and the same results were obtained.

Conclusions
The developed spectrophotometric method is precise, accurate and sensitive.No interference from the frequently encountered excipients and additives.Statistical analysis proves that the method could be applied for the analysis of the studied drugs in their pure forms and in pharmaceutical formulations.

Figure 4 .Figure 5 .
Figure 4. Effect of NaOH concentration on the absorbance of the reaction colored product at 390 nm.

Figure 6 .
Figure 6.Effect of NBD-Cl concentration on the absorbance of the reaction colored product at 390 nm.

1
Scheme 1 Suggested reaction mechanism between sulfide ions and NBD-ClThe production of sulfide ions was confirmed by carrying out specific qualitative tests such as dilute hydrochloric acid, cadmium acetate, sodium nitroprusside and methyelene blue tests[56] or by comparing λ max of the formed chromogen with that obtained after applying the developed method to sodium sulfide and the same results were obtained.

Table 1 .
Chemical structures of the investigated cephalosporin antibiotics.

Table 2 .
Effect of different acids on the absorbance readings of the reaction colored product of cefalexin anhydrous a with NBD-Cl.
a Cefalexin anhydrous concentration used is 15 μg mL -1 ; b Average of three determinations.

Table 3 .
Effect of solvent on λ max and the absorbance of the formed chromogen between cefalexin anhydrous a and NBD-Cl.

Table 4 .
Summary of quantitative parameters and statistical data using the proposed procedure.
a Average of six determinations; b Limit of detection; c Limit of quantitation.

Table 5 .
Accuracy of the proposed method for analysis of the studied drugs at three concentration levels.
a Average of six replicates.

Table 6 .
Intra-and inter-day precision of the proposed spectrophotometric method.
a Average of six determinations.

Table 7 .
Robustness of the proposed spectrophotometric method.
a Theoretical value for t and F at 95% confidence limit, t = 2.228 and F = 5.053; b Reference 48; c Egyptian Pharmaceuticals and chemicals industries Co., S.A.E., Bayad El-Arab, Beni Suef, Egypt; d Pharco Pharmaceuticals, Alexandria under license from Ranbaxy UK; e Bristol-Myers Squibb Pharmaceutical Co., Cairo, Egypt; f Kahira Pharm.& Chem.Ind. Co. under license from Novartis Pharma S.A.E., Cairo, Egypt; g l

Table 9 .
Standard addition method for the assay of the studied drugs in their pharmaceutical dosage forms by the proposed method.

Table 9 (
Continued) a Average of six determinations.