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]. In this study Cefixime and cefoperazone was determined by HPLC method. Several methods have been developed for Cefixime 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].

Macrolides are a group of drugs (typically antibiotics) whose activity stems from the presence of a macrolide ring, a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. The lactone rings are usually 14-, 15-, or 16-membered.

The mechanism of action of macrolides is inhibition of bacterial protein biosynthesis, and they are thought to do this by preventing peptidyltransferase from adding the peptidyl attached to tRNA to the next amino acid [24] (similarly to chloramphenicolas well as inhibiting ribosomal translocation.

From this group we study clarithromycin, several methods have been developed for its determination, including spectrophotometric methods [25-28], high-performance liquid chromatography (HPLC) [29-31] electro chemical methods [32].

In this study, Cefixime, Cefoperazone and Clarithromycin have been determined by RP-HPLC method. The optimized mobile phase was determined as a mixture of methanol : 0.025M potassium dihydrogen phosphate adjusted to pH 7.8 using triethyalamine (20:80, v/v) at a flow rate of 1.0 ml/min. Under these conditions, clarithromycin, cefixime and cefoperazone eluted at 1.59, 2.90, and 4.35 minutes respectively

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.

Clarithromycin (Sigma), Cefoperazone (Sigma), Cefixime (Sigma). Standard solutions 100 µg.ml-1 were 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 (20:80, v/v), filtered and degassed using 0.45µm membrane filter.

2.3. Pharmaceutical preparations:

Ximacef capsuls labeled to contain 400 mg cefixime HCl per capsule. Batch No. 1240009 (Sigma, Egypt).clarihro tablets labeled to contain 250 mg clarithromycin per tablet. Batch No. 502102 (Amriya, Egypt). Cefazone^® vial labeled to contain 500 mg Cefoperazone per vial. Batch No. 00100912 (Pharco, Egypt).

2.4. Procedures:

General chromatographic procedures:Aliquot portions of clarithromycin, cefixime and cefoperazone of 100 µg.ml^-1 ranging from (0.1 – 0.5) ml were transferred into a series of 10-ml flaskes. We take from each flask the same volume into another 10-ml flask and complete 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: Each formulation was powdered and weighed. An accurately amounts of the powder equivalent to 10 mg of each drug were dissolved in 25 ml of methanol , 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 1, 10, 20, 30, 40 and 50 µg.ml^-1 for clarithromycin and cefixime or 2, 10, 20, 30, 40 and 50 for cefoperazone. 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) and it was found to be linear for all drugs.

3. RESULTS AND DISCUSSION:

Optimization of Chromatographic Conditions: All chromatographic conditions are illustrated in table 1. Spectroscopic analysis of the drugs showed that clarithromycin, cefixime and cefoperazone have maximum UV absorbance (λ_max) at 254 nm. The chromatographic detection was performed at 254nm using Surveyor photodiode array detector (PAD) (Thermo Scientific Co. USA). The method was performed on a Hypersil gold^® C18 (10um, 100x4.6mm) column (Thermo Scientific Co. USA). Chromatographic conditions were optimized by changing the mobile phase composition (methanol : 0.025M potassium dihydrogen phosphate buffer ) pH of buffers used in the mobile phase and the flow rate. Different experiments were performed to optimize the mobile phase but adequate separation of drugs could be achieved by altering the composion of mobile phase from (40:60) and (30:70) to (20:80). The optimized mobile phase was determined as a mixture of methanol : 0.025M potassium dihydrogen phosphate adjusted to pH 7.8 using triethyalamine (20:80, v/v) at a flow rate of 1.0 ml/min as shown in (fig. 1).

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

Under these conditions, clarithromycin, cefixime and cefoperazone eluted at 1.59, 2.90, and 4.35 minutes respectively . 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 7.8 .

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 6.5 .

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 7.5 .

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 (40:60, 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 (30:70, 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 (20:80, 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).

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, 35)} where Student’s t-test and F-test were performed for comparison. Reported reference 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 reference 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 .

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 (20:80, 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±5^oC)
pH	7.8

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

Parameters	Clarithromycin *			Cefoperazone *			Cefixime *
Parameters	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	1	1.00	100.31	2	2.03	101.69	1	1.00	100.588
	10	10.04	100.46	10	9.88	98.87	10	9.98	99.845
	20	19.82	99.13	20	19.80	99.04	20	20.17	100.865
	30	30.17	100.59	30	30.33	101.12	30	29.76	99.204
	40	39.95	99.88	40	40.17	100.44	40	39.99	99.981
	50	49.98	99.97	50	49.74	99.49	50	50.08	100.167
Mean			100.062			100.115			100.108
±SD			0.529			1.155			0.584
±RSD			0.528			1.154			0.584
±SE			0.236			0.516			0.261
Variance			0.347			1.555			0.426
Slope			42593.9			81914.2			47759.6
L.D.			0.333			0.666			0.333
L.Q.			1			2			1

* Average of three independent procedures

Table (3). Statistical analysis of results obtained by the proposed method applied on clarithromycin in the form of Clarithro^® tablets compared with reference method.

Parameters	Proposed method	Reported method(34)
N	6	6
Mean	100.234	99.985
±SD	0.787	1.618
±RSD	0.785	1.618
±SE	0.352	0.723
Variance	0.756	2.618
Student-t	0.339(2.02)a
F-test	3.462 (5.05)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 method applied on Cefixime in the form of Ximacef^® capsules compared with reference method.

Parameters	Proposed method	Reported method(35)
N	6	6
Mean	100.075	99.616
±SD	1.063	1.445
±RSD	1.062	1.450
±SE	0.475	0.646
Variance	1.312	2.088
Student-t	0.627 (2.02)a
F-test	1.591 (5.05)b

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

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 (5). Statistical analysis of results obtained by the proposed method applied on cefoperazone in the form of Cefazone^® vials compared with reference method

Parameters	Proposed method	Reported method(35)
N	6	6
Mean	100.262	99.729
±SD	0.851	1.001
±RSD	0.849	1.004
±SE	0.380	0.448
Variance	0.904	1.003
Student-t	0.994 (2.02)a
F-test	1.109 (5.05)b

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

Table(6). Intra – day and interday precision of three drugs

Drug	Concentration µg/ml	Intraday		Interday
Drug	Concentration µg/ml	mean± SD	RSD	mean± SD	RSD
Clarithromycin	50	100.9 ± 0.675	0.68	100.1± 0.477	0.476
Cefixime	50	101.28 ± 0.748	0.75	99.8 ± 0.46	0.45
Cefoperazone	50	99.56 ± 0.98	0.98	101.32± 0.38	0.38

Table (7). Robustness of the method

Parameters	% of recovery ± SD
Parameters	Clarithromycin	Cefixime	Cefoperazone
Flow rate 0.9	98.87±1.44	98.12±1.56	101.8±0.99
Flow rate 1.1	98.5±1.7	99.68±1.32	101.5±0.15
Mobile phase19:81	101.8±0.82	101.7±0.93	101.7±0.17
Mobile phase 21:79	98.38±1.2	99.06±1.4	98.5±1.8
pH 7.7	101.1±0.43	99.08±1.4	100.32±1.36
pH 7.9	100.9±1.29	99.6±1.6	98.3±1.47

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.

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Received on 09.11.2014 Accepted on 25.11.2014

Asian J. Pharm. Res. 4(4): Oct.-Dec.2014; Page 180-185

Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazige University, Zagazig, Egypt.

*Corresponding Author E-mail: elmohands_eg@yahoo.com