1. INTRODUCTION:

Cephalosporins, like all β-lactam antibiotics, inhibit bacterial growth by interfering with a specific step in bacterial cell wall synthesis (1). Cephalosporins consist of a fused β-lactam-A -dihydrothiazine two-ring system, known as 7-amino cephalosporanic acid (7-ACA) and vary in their side chain substituent at C₃(R₂), and C₇ (acylamido, R1) (2). In this study Cefixime was determined spectrophotometically. Several methods have been developed for its determination, including spectrophotometric methods (6-3), high-performance liquid chromatography (HPLC) (11-7), Electro chemical methods (13-12).

Several methods have been developed for Cefotaxime determination, including spectrophotometric methods (14-20), high-performance liquid chromatography (HPLC)(21-26), Electro chemical methods (^.27).

Many spectrophotometric methods were developed for cephalosporins determinations that based on hydrolysis of these drugs using alkaline degradation and subsequent reaction of the formed sulphide ions with chromogenic reagents (28). Ellman's reagent (DTNB) has been reported as reagent for determination of amines, SH- and SS- group determination in proteins (29) and for spectrophotometric determination of many compound.

Ellman's reagent can be used for measuring low-molecular weight thiols such as glutathione in both pure solutions and biological samples, such as blood. (30)

On the basis of the aforementioned reasons, it was thought to use this reagent for a quantitative spectrophotometric determination of cefotaxime Na and cefixime. The developed method is based on the alkaline hydrolysis of the studied drugs and subsequent reactions of the resulting hydrolysates with DTNB as a chromogenic reagent which could be used for their analysis in pure forms and in pharmaceutical formulations. This method is selective for cephalosporins since other B-lactam antibiotics such as penicillins do not give sulphide ions under the degradation conditions employed (31-32).

2. EXPERIMENTAL:

2.1. Apparatus:

Labomed^®Spectro UV-VIS Double Beam (UVD-2950) Spectrophotometer with matched 1 cm quartz cells connected to windows compatible computer using UV Win 5 Software v5.0.5. Spectronic Genesys^® UV-VIS Spectrophotometer connected to an IBM PC computer loaded with FLWINLAB software. Consort P400^® digital pH-meter for pH adjustment.

2.2. Materials and reagents:

All solvents and reagents were of analytical grade and double distilled water was used throughout the work. Cefixime (Sigma-Egypt) Standard stock solutions 100 µg.ml^-1 of cefixim was prepared by dissolving pure drug in 25 ml methanol, then completed to 100 ml with bidistilled water). Standard working solutions 4µg.ml^-1 of cefixime )was prepared by taking 4 ml from stock solution and complete to 100 ml with bidistilled water). Cefotaxime Na (Eipico -Egypt) Standard stock solutions 100 µg.ml^- of cefotaxime was prepared by dissolving pure drug in 100 ml bidistilled water. Standard working solutions 8µg.ml^{- 1 )was} prepared by taking 8 ml from stock solution and complete to 100 ml with bidistilled water). 5,5'-dithiobis(2-nitrobenzoic acid)DTNB (Aldrich Chemical Co. Ltd., Dorset, England freshly prepared 2.5x 10^-4 M in 0.15M phosphate buffer pH 7.2. Sodium hydroxide (El Nasr Chemical Co. Cairo Egypt) 0.5M aqueous solution.

2.3. Pharmaceutical preparations:

The following available preparations were analyzed

Ximacef^® capsule labeled to contain 400 mg cefixime per capsule. Batch No. 1240009 (Sigma, Egypt). Cefotax^® vial labeled to contain 250 mg cefotaxime Na per vial. Batch No. 1204846 (Eipico, Egypt).

2.4. General spectrophotometric procedures and construction of calibration curves using DTNB method:

Aliquot portions of the standard working solutions (0.1-0.5 ml) for the two drugs was transferred into 10-ml volumetric flask. 0.5 ml of 0.5 M NaOH Was added and completed to volume with bi-distilled water then put this content in test tube that are heated in water bath at 90^oC for 25 min then cooled in tap water and left to take room temperature. One milliliter of the resulting drug hydrolysate was pipetted into 10ml volumetric flask 1.0 ml of 2.5x10^-4 M DTNB was added .The resulting solution was mixed well and the flask was completed to volume with bi-distilled water. The absorbance was measured at 411 nm for cefixime and 417nm for cefotaxime against reagent blank that treated similarly.

2.4.3. Procedures for pharmaceutical preparation:

For Ximacef capsule: 10 capsules were weighed and powdered. An accurately amounts of the powder equivalent to 10.8 mg of cefixime were dissolved in 25 ml of Methanol, filtered into 100-ml measuring flask and completed to volume with bidistilled water to 100 ml .From the stock solution we make the standard working solution and complete the procedures as previously mentioned under materials and reagents and the general procedures. For Cefotax vials, contents of one vial were weighed. An accurate amounts of the vial powder equivalent to 11.3 mg of cefotaxime sodium were dissolved in bidistilled water in 100- measuring flask and completed to volume with bidistilled water to 100ml .From the stock solution we make the standard working solution and complete the procedures as previously mentioned under materials and reagents and the general procedures.

3. RESULTS AND DISCUSSION:

Some thiocompounds were previously reported to produce sulphide ions upon alkaline degradation and it was found to be one of their major degradation product^[148-149]. In the proposed method thiols react with this compound, cleaving the disulfide bond to give 2-nitro-5-thiobenzoate (TNB^-), which ionizes to the TNB^2- dianion in water at neutral and alkaline pH. This TNB^2- ion has a yellow color(31)This reaction is rapid and stoichiometric, with the addition of one mole of thiol releasing one mole of TNB. The TNB^2- is quantified in a spectrophotometer by measuring the absorbance of yellow – coloured chromophore (λ max at 411nm for cefixime or 417 nm for cefotaxime). The proposed reaction mechanism is given as following (32).

All parameters were studied as follow:

3.1.-Absorption spectra:

The absorption spectrum of coloured product of cefixime and cefotaxime Na hydrolysates through the interaction with DTNB shows absorption maximum at 411 nm and 417nm respectively (Fig.2).

Figure2. Absorption spectra of reaction product of cefixime (A) and cefotaxime (B) with DTNB after hydrolysis by NaOH at maximum wave length 411 nm. and 417nm respectively.

3.2. Effect of NaOH concentration:

The influence of sodium hydroxide concentration on producing the maximum absorption intensity was investigated using different volumes of 0.5 M NaOH. The best volume was selected for further work (Fig.3) .

Figure 3. Effect of NaOH concentration on the absorbance of the reaction coloured product at 411 nm (cefixime) or 417nm (cefotaxime ).

3.3. Effect of hydrolysis time:

The effect of hydrolysis time on the absorption intensity was studied using different heating times in water bath at 90^oC starting from 10 min to 1 hour. The maximum absorption intensity was found to be 25 min and remained stable for at least 60 min. as shown in (Fig 4)

3.4. Effect of DTNB concentration:

The volume of 2.5x 10^-4DTNB for the maximum colour development was varied in the range of 0.2 – 1.8 ml. It was found that 1 ml of DTNB was the most suitable volume for determination as shown in (Fig.5) .

Figure 4. Effect of hydrolysis time on the absorbance of the reaction colored product at 411nm (cefixime) or 417nm (cefotaxime).

Fig. 5-Effect of 2.5 X 10^-4 M DTNB volume on the absorbance of the reaction colored product at 411 nm (cefixime) or 417nm (cefotaxime).

3.5. Effect of reaction time:

The reaction between the investigated drug hydrolysates and DTNB was very rapid but the interaction colored product can survive before dilution unchanged only for 15 minutes.

3.6. Effect of temperature:

The effect of temperature on the absorption intensity was studied using different temperatures in a water bath (25^oC–100^oC) and the reaction was carried out as usual. The obtained absorbance readings were plotted against temperature. The maximum absorption intensity was attained at 90 ^oC as shown in (Fig.6).

4. METHOD VALIDATION:

The developed methods were validated according to international conference on harmonization guidelines (33).The linearity range of absorbance as a function of drug concentration (Table 1) provides good indication about sensitivity of reagents used.

Figure 6. Effect of temperature on the absorbance of the reaction colored product at 411 nm (cefixime)or 417nm (cefotaxime).

Calibration curves have correlation coefficients (r)=0.999 indicating good linearity. The accuracy of the methods were determined by investigating the recovery of drugs at concentration levels covering the specified range (three replicates of each concentration). The results showed excellent recoveries (table 2). Also, the Limit of detection (L.D.), Limit of quantitation (L.Q.), Sandell’s sensitivity (S.S.) and Molar absorptivity were calculated. The small values of SD and % RSD point to high precision of the proposed method. Intra - day precision was evaluated by calculating standard deviation (SD) of five replicate determinations using the same solution containing pure drugs (table 6). The SD values revealed the high precision of the methods. For inter - day reproducibility on a day - to - day basis, a series was run, in which the standard drug solutions were analyzed each for five days (table 6). Robustness was examined by evaluating the influence of small variation of method variables including NaOH concentration, DTNB concentration, heating temperature and heating time on the method suitability and sensitivity. (Table 7) shows that none of these variables significantly affects the performance of the method which indicates robustness of the proposed method.

Table (1). Analytical parameters for the determination of cefixime and cefotaxime Na by DTNB..

Cefixime	Cefotaxime	PARAMETERS
411	417	Wave length , nm
1	1	Volume of DTNB, ml
0.5	0.5	Volume of NaOH (0.5 M), ml
25	25	Time of hydrolysis min.
90	90	Temperature ^oC
0.4-2	0.8- 4	Beer's law limits, µg/ml
y=0.275x+ 0.161	y=0.194x+ 0.093	Regression equation
0.999	0.999	Correlation Coefficient

y = a + bx, where y is the absorbance, a is the intercept, b is the slope and x is the concentration in μg/ml.

Table (2) Determination of cefixime and cefotaxime Na using DTNB method.

Parameters	DTNB
	Cefotaxime			Cefixime
	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	0.8	0.804	100.515	0.4	0.403	100.909
	1.6	1.613	100.837	0.8	0.810	101.363
	2.4	2.381	99.226	1.2	1.196	99.696
	3.2	3.206	100.193	1.6	1.6	100
	4	4..010	100.257	2	2.007	100.363
Mean			100.206			100.34
±SD			0.603			0.674
±RSD			0.602			0.673
±SE			0.269			0.301
Variance			0.364			0.455
Slope			0.194			0.275
L.D.			0.26			0.13
L.Q.			0.77			0.38
S.S.			0.004			0.001

y = a + bx, where y is the absorbance, a is the intercept, b is the slope and x is the concentration in µg/ml.

Table (3) The proposed methods applied on cefixime in the form of Ximacef® capsules compared with reference method.

Parameters	Methylene Blue method	Methyl Orange method	Reported method (24)
N	5	5	5
Mean	100.64	100.56	99.84
S D	0.264	0.357	1.226
RSD	0.514	0.598	1.226
SE	0.510	0.594	0.550
Variance	0.229	0.267	1.051
Student-t	1.423 (2.57)^a	0.903 (2.57)^a
F-test	4.58(6.256)^b	3.93(6.256)^b

a and b are the Theoretical Student t-values and F-ratios at p=0.05.

Table (4). Statistical analysis of results obtained by the proposed methods applied on Claritromycin in the Clarithro^® tablets compared with reference method.

Parameters	Proposed method	Reported method(24)
N	5	5
Mean	100.409	99.99
S D	0.532	0.851
RSD	0.530	0.851
SE	0.238	0.383
Variance	0.283	0.735
Student-t	0.931(2.57) a
F-test	2.597(6.256) b

a and b are the Theoretical Student t-values and F-ratios at p=0.05.

5. APPLICATIONS:

Some Pharmaceutical formulations containing the stated drugs have been successfully analyzed by the proposed methods. The reported reference method is based on the alkaline hydrolysis of the drugs and subsequent reactions of the resulting hydrolysates with NBD-Cl (4-Chloro-7-Nitro-2,1,3-BenzoxaDiazole) as a chromogenic reagent and detection at 401nm Results obtained from proposed methods were compared to those obtained by applying reported reference methods for cefixime (34) and cefotaxime Na (34).where Student’s t-test and F-ratio were performed for comparison. Results are shown in (table 4) where the calculated t and F values were less than tabulated values which in turn indicate that there is no significant difference between proposed methods and reference ones relative to accuracy and precision.

6. Conclusion:

Spectrophotometry is simple and inexpensive. The proposed methods require sodium hydroxide and DTNB as reagents which are readily available, and pH adjustment is required and the procedures do not involve any critical reaction conditions or tedious sample preparation. Moreover, methods are simple, moderately fast, accurate and adequately sensitive. The recovery % obtained by the proposed methods was between 99.2% and 101.3%, within the acceptance level 95% to 105%. The present methods are superior to the reference method with respect to both sensitivity and selectivity. The methods have been successfully applied for the analysis of marketed cefotaxime Na vials and cefixime capsules.

Table (5). The proposed methods applied on cefotaxime in Cefotax^® vials compared with reference method..

Parameters	Proposed method	Reported method(24)
N	5	5
Mean	100.161	99.75
S D	0.735	1.620
RSD	0.734	1.618
SE	0.329	0.724
Variance	0.541	1.450
Student-t	0.517(2.57) a
F-test	2.680(6.256) b

a and b are the Theoretical Student t-values and F-ratios at p=0.05.

Table (6). Intraday and interday precision for the determination of cefixime and cefotaxime Na using DTNB method.

Intraday and interday precision
Drug	Intraday		Interday
Drug	mean of recovery ±SD	RSD	mean of recovery ±SD	RSD
Cefixime	100.01±0.577	0.577	100.21±0.878	0.877
Cefotaxime Na	100.02±0.599	0.599	100.9±0.71	0.71

Table (7). Robustness for the determination of cefixime, and cefotaxime Na using DTNB method.

Robustness
Parameters	Cefixime	Parameters	Cefotaxime Na
Parameters	mean of recovery ±SD	Parameters	mean of recovery ±SD
NaOH		NaOH
0.45M	98.2±1.04	0.45M	101.4±0.476
0.55M	101.6±1.18	0.55M	100.19±1.21
DTNB		DTNB
0.95ml	100.6±0.69	0.95ml	99.06±1.4
1.05ml	99.05±0.69	1.05ml	100±0.92
Temperature		Temperature
85^oC	100±0.92	85^oC	101.3±0.13
95˚C	98.14±0.19	95 ˚C	101.8±0.51
Time		Time
20min.	101.92±0.77	20min.	98.57±1.56
30min.	101.47±52	30min.	101.5±1.35

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Received on 09.11.2014 Accepted on 26.11.2014

Asian J. Res. Pharm. Sci. 4(4): Oct.-Dec. 2014; Page 196-201