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 substituents at C₃(R₂), and C₇ (acylamido, R1) [2]. Several methods have been developed for Cefepime determination, including spectrophotometric methds [3-6], high-performance liquid chromatography (HPLC) [7-11] ^, Electro chemical methods [12-13] . Several methods have been developed for Cefoperazone determination, including spectrophotometric methds [14-16], high-performance liquid chromatography (HPLC) [17-21]^, Electro chemical methods [22-23].

Several methods have been developed for Cefotaxime determination, including specrophotometric methds [24-28], high- performance liquid chromatography (HPLC) [29-31] electro chemical methods [32].

2. Experimental:

2.1. Apparatus:

High performance liquid chromatography (HPLC) apparatus equipped with a Surveyor quaternary pump with Intel vacuum degasser (Thermo Scientific Co. USA) , Surveyor autosampler plus (Thermo Scientific Co., USA), Surveyor photodiode array detector (PAD) (Thermo Scientific Co. USA).

Computer with a software chromo quest 5 (Surveyor Thermo Scientific Co. USA), for data collection and analysis, Hypersil gold C18 (10um, 100x4.6mm) column (Thermo Scientific Co. USA). Autosampler vials 1.8 ml screw cap, Thermo Scientific, USA.Consort P400^® digital pH-meter for pH adjustment.

2.2. Materials and reagents:

All solvents and reagents were of an HPLC analytical grade (methanol, potassium dihydrogen phosphate and triethyalamine) were supported from Romil, England.Cefepime HCl (Adwia, Egypt), Cefoperazone (Sigma, Egypt), Cefotaxime Na (Eipico, Egypt). Standard solutions 100 µg.ml^-1were prepared individually by dissolving 10 mg of each pure drug in 100 ml of the mobile phase. Mobile phase was a freshly prepared binary mixture of methanol: 0.025M potassium dihydrogen phosphate buffer adjusted to pH 7.8 using triethyalamine (18:82, v/v), filtered and degassed using 0.45µm membrane filter.

2.3. Pharmaceutical preparations:

WinCef vial labeled to contain 500 mg cefepime HCl per vial. Batch No. 62230029 (Adwia, Egypt).

Cefazone^® vial labeled to contain 500 mg cefoperazone per vial. Batch No. 00100912 (Pharco, Egypt).

Cefotax^® vial labeled to contain250 mg cefotaxime Na per vial. Batch No. 1204846 (Eipico, Egypt).

2.4. Procedures:

General chromatographic procedure:

Aliquot portions of cefepime, cefoperazone and cefotaxime of 100 µg.ml^-1 ranging from (0.1 – 0.5 ml) were transferred into a 10-ml flask and completed to 10 ml with the mobile phase and this is the mixture of the three drugs. This mixture was measured at 254 nm.. Sample preparation: Three vial of each formulation was weighed. An accurate weight of the powder equivalent to 10 mg of each drug were dissolved in 25 ml of the mobile phase, filtered into 100 - ml measuring flask and completed to volume with the mobile phase. The procedure was then completed as previously mentioned under the general procedure. Construction of calibration curves: Appropriate mixed dilutions of the standard stock solutions were done in 10 - ml volumetric flasks to get final concentrations of 10, 20, 30, 40 and 50 µg.ml^-1 for the three drugs. A 10 μl of each mixture was injected into the column and the chromatogram was measured at 254 nm. A graph was plotted as concentration of each drug against response (peak area).

3. Results and discussion:

All chromatographic conditions are illustrated in table 8. Spectroscopic analysis of the drugs showed that cefepime HCl, cefoperazone, and cefotaxime Na have maximum UV absorbance (λ_max) at 254 nm. The chromatographic detection was performed at 275-254-and 210 nm using a UV – Visible detector. The method was performed on a Hypersil gold C18 (10um, 100x4.6mm) supported from (Merck, Germany) Chromatographic conditions were optimized by changing the mobile phase composition (binary mixture of methanol: 0.025M potassium dihydrogen phosphate buffer) and pH of buffer used in the mobile phase and studying the absorbion at 210,254, and 275 nm applied. Different experiments were performed to optimize the mobile phase but adequate separation of drugs could be achieved by altering the composition of mobile phase from (22:78), (20:80) to (18:82)a good separation was achieved at 254nm (fig. 6 and 7,8 ) The optimized mobile phase was determined as a mixture of methanol : 0.025M potassium dihydrogen phosphate adjusted to pH 7.8 using trimethyalamine (18:82, v/v) at a flow rate of 1.0 ml/min. Under these conditions, cefepime HCl, cefotaxime, and cefoperazone eluted at 1.815, 5.635, and 14.592 minutes respectively with a run time of 15 minutes. as shown in (fig. 1).

Figure 1.HPLC Chromatogram of authentic mixture of 50 µg.ml^-1clarithromycin at (1.5 min), cefixime at(2.90 min) and cefoperazone at (4.35min).

A typical chromatogram for simultaneous estimation of the three drugs in their pharmaceutical dosage forms obtained by using the aforementioned mobile phase is illustrated in (fig. 2).

Figure 2. HPLC Chromatogram of the mixture of 50 µg.ml^-1clarithromycin in Clarithro^® tablets at (1.5 min) , cefixime in Ximacef^® capsules at(2.90 min) and cefoperazone in cefazone^® vials at(4.35min).

All parameter were studied as follow:

3.1.Effect of pH of the buffer:

Figure 3. HPLC Chromatogram of authentic mixture of 50 µg.ml-1 clarithromycin, cefixime and cefoperazone respectively

Column: Hypersil gold C18 (10um, 100x4.6mm) column at pH 7 .

Figure 4. HPLC Chromatogram of authentic mixture of 50 µg.ml-1 clarithromycin, cefixime and cefoperazone respectively

Column: Hypersil gold C18 (10um, 100x4.6mm) column at pH 7.8 .

Figure 5. HPLC Chromatogram of authentic mixture of 50 µg.ml-1 clarithromycin, cefixime and cefoperazone respectively

Column: Hypersil gold C18 (10um, 100x4.6mm) column at pH 8 .

3.2.Effect of mobile phase composion:

Figure 6.HPLC Chromatogram of authentic mixture of 50 µg.ml^-1 clarithromycin ,cefixime and cefoperazone respectively at Mobile phase : MeOH : 0.025M KH₂PO₄ and pH 7.8

(20:80, v/v) .

Figure 7.HPLC Chromatogram of authentic mixture of 50 µg.ml^-1 clarithromycin ,cefixime and cefoperazone respectively at Mobile phase : MeOH : 0.025M KH₂PO₄ and pH 7.8

(22:78, v/v) .

Figure 8.HPLC Chromatogram of authentic mixture of 50 µg.ml^-1 clarithromycin ,cefixime and cefoperazone respectively at Mobile phase : MeOH : 0.025M KH₂PO₄ and pH 7.8

(18:82, v/v) .

3.3.Effect of flow rate :

Figure 9. HPLC Chromatogram of authentic mixture of clarithromycin, cefixime and cefoperazone respectively at flow rate 0.8 ml\min

Figure 10. HPLC Chromatogram of authentic mixture of clarithromycin, cefixime and cefoperazone respectively at flow rate 1.4 ml\min

Figure 11. HPLC Chromatogram of authentic mixture of clarithromycin, cefixime and cefoperazone respectively at flow rate 1 ml\min

4. METHOD VALIDATION:

The developed methods were validated according to international conference on harmonization guidelines (33). Calibration curves have correlation coefficients (r) higher than 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 tables (3), (4). Also, the Limit of detection (L.D.), Limit of quantitation (L.Q.), Sandell’s sensitivity (S.S.) and Molar absorbitivity were calculated. Intraday precision was evaluated by calculating standard deviation (SD) of five replicate determinations using the same solution containing pure drug table( 8),(9). For interday reproducibility on a day - to - day basis, a series was run, in which the standard drug solutions were analyzed each for five days. The day - to - day SD values were shown in table (8) ,(9). The robustness of the methods was evaluated by making small changes in the volume of acid , dye volume and bromated bromine volume where the effect of the changes was studied on the percent recovery of drugs. Table (10), (11).

Table (1). Chromatographic Conditions for the proposed method.

Parameters	Conditions
Column	Hypersil gold C18 (10um, 100x4.6mm) column
Mobile phase	Isocratic binary mobile phase of MeOH : 0.025M KH2PO4 adjusted to pH 7.8 using triethyl amine (18:82, v/v), filtered and degassed using 0.45µm membrane filter
UV detection, nm	254
Flow rate, ml/min	1
Injected volume, µl	10
Pressure, psig	11
Temperature	Ambient (25±5oC)
pH	7.8

Table (2). Results of the analysis for the proposed method

Parameters	Cefepime HCl *			Cefotaxime Na*			Cefoperazon*
Parameters	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	10	10.14	101.4	10	9.91	99.1	10	9.98	99.8
	20	19.66	98.3	20	19.81	99.05	20	20.17	100.85
	30	30.46	101.53	30	30.33	101.1	30	29.76	99.2
	40	39.5	98.75	40	40.17	100.425	40	39.99	99.97
	50	50.21	100.42	50	49.74	99.48	50	50.08	100.16
Mean			100.08			99.831			100.02
±SD			1.491			0.898			0.599
±RSD			1.489			0.899			0.598
±SE			0.666			0.401			0.267
Variance			2.224			0.807			0.359
Slope			42681			81302			47892
L.D.			3.3			3.33			3.3
L.Q.			9.9			10			9.9

* Average of three independent procedures

Table (3). The proposed method applied on cefepime in Winecef^®vials compared with reported method.

Parameters	Proposed method	Reported method(34)
N	5	5
Mean	100.014	99.90
±SD	0.579	1.13
±RSD	0.578	1.13
±SE	0.258	0.340
Variance	0.336	1.278
Student-t	0.201 (2.57) a
F-test	3.803 (6.256) b

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

Table (4) The proposed method applied on cefotaxime in Cefotax® vials compared with reported method.

Parameters	Proposed method	Reported method(34)
N	5	5
Mean	100.19	99.64
±SD	0.879	1.806
±RSD	0.877	1.810
±SE	0.393	0.730
Variance	0.773	1.520
Student-t	0.614(2.57) a
F-test	1.966 (6.256) b

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

Table (5). The proposed method applied on cefoperazone in Cefazone® vials compared with reported method.

Parameters	Proposed method	Reported method(35)
N	5	6
Mean	100.069	99.729
±SD	0.552	1.001
±RSD	0.552	1.004
±SE	0.247	0.448
Variance	0.305	1.003
Student-t	0.675 (2.262) a
F-test	3.288 (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 stated drugs have been successfully analyzed by the proposed method. Results obtained were compared to those obtained by applying reported reference methods⁽³⁴⁾ where Student’s t-test and F-test were performed for comparison. Reported refrence method in case of cefixime and cefoperazone use tetrabutylammonium hydroxide buffer at pH 6.8 and acetonitrile and measure the compounds at 254 nm . Reported refrence method for clarithromycin use potassium dihydrogen phosphate buffer at pH4.4 and measuring at 205 nm with flow rate 1.1 ml\min . Results are shown in tables 3, 4 and 5 where the calculated t and F values were less than tabulated values for the three drugs which in turn indicate that there is no significant difference between proposed method and reference ones relative to accuracy and precision .

6. Conclusion:

An RP-HPLC method for rapid simultaneous estimation of clarithromycin, cefixime and cefoperazone within less than 5 minutes was developed and validated. The results obtained indicate that the proposed method is rapid, accurate, selective, and reproducible. Linearity was observed over a concentration range of 1 to 50 ug.ml^-1 for cefixime and clarithromycin and in the range of 2 to 50 ug.ml^-1 for cefoperazone . The method has been successfully applied for the analysis of marketed formulations. It can be used for the routine analysis of formulations containing any one of the above drugs or their combinations without any alteration in the assay.

Table(6 )Intraday and interday precision for HPLC determination of cefepime, cefotaxime and cefoperazone in bulk and dosage form .

Drug	conc. µg/ml	Intraday		Interday
Drug	conc. µg/ml	mean± SD	RSD	mean± SD	RSD
Cefepime	50	100.01 ± 0.577	0.577	101.4± 0.476	0.479
Cefotaxime	50	100.02 ± 0.599	0.599	99.56 ± 0.98	0.98
Cefoperazone	50	100.21 ± 0.878	0.877	100.19± 1.21	1.21

Table (7). Robustness for HPLC determination of cefepime, cefotaxime and cefoperazone in bulk and dosage form .

Parameters	% of recovery ± SD
Parameters	Cefepime	Cefepime	Cefepime
Flow rate 0.9	98.7±1.4	98.7±1.4	98.7±1.4
Flow rate 1.1	101.6±1.18	101.6±1.18	101.6±1.18
Mobile phase 17:83	99±0.69	99±0.69	99±0.69
Mobile phase 19:81	100.6±0.69	100.6±0.69	100.6±0.69
pH 7.7	101.1±0.43	101.1±0.43	101.1±0.43
pH 7.9	99.05±0.69	99.05±0.69	99.05±0.69

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Received on 09.11.2014 Accepted on 28.11.2014

Asian J. Pharm. Ana. 4(4): Oct. - Dec. 2014; Page 168-173