1. INTRODUCTION:

Fluoroquinolones are a class of compounds that comprise a large and expanding group of synthetic antimicrobial agents. Structurally, all fluoroquinolones contain a fluorine atom at the 6-position of the basic quinolone nucleus. Despite the basic similarity in the core structure of these molecules, their physicochemical properties, pharmacokinetic characteristics and microbial activities can vary markedly across compounds¹.

Quinolones act by inhibiting the activities of DNA gyrase (enzyme catalyzing changes in the degree of double-stranded DNA supercoiling) in gram-negative bacteria, which in turn inhibit replication and transcription of bacterial DNA. Prevention of DNA synthesis ultimately results in rapid cell death. This unique mechanism of action may account for the low rate of cross-resistance with other antimicrobial classes².

Quinolones similarly inhibit the in vitro activities of DNA topoisomerase IV (enzyme mediating relaxation of duplex DNA and the unlinking of daughter chromosomes following replication) which is believed to be the primary target in gram-positive bacteria³.

Levofloxacin HCL ( (-)-(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6- carboxylic acid Hydrochloride), Lomefloxacin HCl ( (±)-1-ethyl-6, 8-difluoro-1,4-dihydro-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid Hydrochloride), Gatifloxacin ((±)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid) and Sparfloxacin (5-Amino-1-cyclopropyl-7-(cis-3,5-dimethyl-1-piperazinyl)-6,8-difluoro-1,4 dihydro-4-oxo-3-quinolinecarboxylic acid)⁴ are fluoroquinolones and antimicrobials with potent activity against a broad spectrum of bacteria.

Several HPLC methods had been developed for determination of these drugs individually^5-15 or in combination with other drugs^16-21 but No HPLC method for simultaneous estimation of these four drugs using monolithic silica columns has been reported till date.

In the present study, an attempt has been made to develop a method for the simultaneous estimation of levofloxacin HCl, lomefloxacin HCl, gatifloxacin and sparfloxacin. It can also be applied for routine analysis of either one or of any combinations of these drugs in dosage forms.

2. EXPERIMENTAL:

2.1. Apparatus:

· Waters 2487^® HPLC instrument (U.S.A) with Waters automated gradient controller, Chromolith^® Performance RP-18e column (100 x 4.6 mm), dual λ absorbance detector, binary 515 HPLC pumps and connected to PC computer loaded with Millenium 32 software.

· 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 ortho - phosphoric acid were supported from Romil, England).

· Levofloxacin HCl (Pharaonia), Lomefloxacin HCl (Sigma), Gatifloxacin (EPCI) and Sparfloxacin (Global Napi). Standard solutions 400 µg.ml^-1were prepared individually by dissolving 40 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 adjusted to pH 3 using ortho - phosphoric acid (20:80, v/v), filtered and degassed using 0.45µm membrane filter.

· Manufacturing impurities like Benzyl amine, Ethylene diamine, N-methyl piperazine, 2,3,4-trifluoroaniline, 2,6-dimethyl piperazine, Methyl chloroacetate and Diethyl malonate were supported from Merck, Germany.

2.3. Pharmaceutical preparations:

The following available pharmaceutical preparations were analyzed

· Leeflox^® tablets labeled to contain 500 mg levofloxacin HCl per tablet. Batch No.1149004 (Pharaonia, Egypt).

· Lomex^® tablets labeled to contain 400 mg lomefloxacin HCl per tablet. Batch No. 4002204 (Sigma, Egypt).

· Gatiflox^® tablets labeled to contain 400 mg gatifloxacin per tablet. Batch No. 171080310 (EPCI, Egypt).

· Spara^® tablets labeled to contain 200 mg sparfloxaacin per tablet. Batch No. 911601 (Global Napi, Egypt).

2.4. Procedures:

2.4.1. Preparation of calibration curves:

Appropriate mixed dilutions of the standard stock solutions were done in 10 - ml volumetric flasks to get a final concentrations of 1, 10, 20, 40, 60 and 80 µg.ml^-1 for all drugs. A 10 μl of each mixture was injected into the column and the chromatogram was obtained at 290 nm. A graph was plotted as concentration of drugs against response (peak area) and it was found to be linear for all drugs.

2.4.2. Sample preparation:

10 tablets of each formulation were weighed and crushed. An accurately amounts of the powder equivalent to 40 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 mentioned above under the general procedure.

3. RESULTS AND DISCUSSION:

Monolithic silica columns were first introduced in 1991 by Minakuchi and Soga²². The preparation of these silica rod materials involved a sol-gel process using highly pure silica. The formed silica rod is then encased in poly ether ethyl ketone shrink-warp tubing, which prevents void formation. The obtained highly porous skeleton is characterized by a bimodal pore structure consisting of large macropores (diameter 2 µm) and mesopores (13 nm in diameter). The large macropores are responsible for a low flow resistance and therefore allow for the application of high eluent flow rates, while the small pores ensure sufficient surface area (300 m²/g approximately) for separation efficiency. As aresult, High flow rates could be used with monolithic columns due to the high porosity of the column provided mainly with macropores. Besides, high efficiency is ensured by the mesopores that provide very large surface area for separation²³ (Fig. 1).

Fig.(1) Monolithic Silica Skeleton A, Macropores and Mesopores B.

The difference between monolithic and conventional particle-packed columns is shown in Figure 2.

Conventional Silica "Particle-Based"

High flow resistance:

Limits ability to shorten run times.

High backpressure:

Reduces life of system.

Monolithic porous silica rod

High flow rates:

Significantly shorter run times.

Low backpressures:

Less stress on system.

Fig.(2) Representative conventional particle-packed vs. monolithic silica HPLC columns.

Furthermore, the separation efficiency of monolithic columns does not decrease significantly when the flow rate is increased as in case of particulate columns. Accordingly, it is possible to operate monolithic columns at high flow rates with minimal loss of peak resolution. High resistance to blockage and long column life time are also advantages of high porosity²⁴.

3.1. Optimization of Chromatographic Conditions:

All chromatographic conditions are illustrated in table 1. Spectroscopic analysis of the drugs showed that levofloxacin HCl, lomefloxacin HCl, gatifloxacin and sparfloxacin have maximum UV absorbance (λ_max) at 293 nm, 287 nm, 290 nm, and 291 nm respectively. Therefore, the chromatographic detection was performed at 290 nm using a UV – Visible detector. The method was performed on a Chromolith^® Performance RP-18e (100 x 4.6 mm) supported from Germany. It was observed that when a combination of all the four drugs was injected, levofloxacin HCl, lomefloxacin HCl together gave a mixed peak due to their polar nature. Chromatographic conditions were optimized by changing the mobile phase composition and buffers used in the mobile phase. Different experiments were performed to optimize the mobile phase but adequate separation of drugs could not be achieved. By altering the pH of mobile phase from 4.5 to 3 a good separation was achieved (fig. 3 and 4). The optimized mobile phase was determined as a mixture of methanol: 0.025M potassium dihydrogen phosphate adjusted to pH 3 using ortho- phosphoric acid (20:80, v/v) at a flow rate of 4.0 ml/min. Under these conditions, levofloxacin HCl, lomefloxacin HCl, gatifloxacin and sparfloxacin were eluted at 1.11, 2, 2.98, and 4.3 minutes respectively with a run time of 10 minutes.

A. B.

Fig.(3) HPLC Chromatogram of authentic mixture of levofloxacin HCl (v), lomefloxacin HCl (m) , gatifloxacin (g) and sparfloxacin (s).

Column : Chromolith^® Performance RP-18e (100 x 4.6 mm).

Mobile phase : MeOH : 0.025M KH₂PO₄ adjusted to pH 4.5 using

ortho phosphoric acid (20:80, v/v).

Flow rate : 4 ml/min.

pH : 4.5(A) and 4(B).

A B

Fig.(4) HPLC Chromatogram of authentic mixture of levofloxacin HCl (v), lomefloxacin HCl (m) , gatifloxacin (g) and sparfloxacin (s).

Column : Chromolith^® Performance RP-18e (100 x 4.6 mm).

Mobile phase : MeOH : 0.025M KH₂PO₄ adjusted to pH 3.5 using

ortho phosphoric acid (20:80, v/v).

Flow rate : 4 ml/min.

pH : 3.5(A) and 3(B).

A typical chromatogram for simultaneous estimation of the four drugs obtained by using the aforementioned mobile phase is illustrated in figures 4 (authentic mixture) and 5 (tablet formulations).

Also, chromatographic conditions were appropriate for separation of each drug from its manufacturing impurities. N-methyl piperazine and methyl chloroacetate²⁵ can be separated from levofloxacin HCl and eluted at 0.72 and 2.9 minutes, respectively (fig. 6). 2,3,4-trifluoroaniline²⁶ can be separated from lomefloxacin HCl and eluted at 1.19 minutes (Fig. 7). Ethylene diamine and 2,3,4-trifluoroaniline²⁷ can be separated from gatifloxacin and eluted at 0.53 and 1.19 minutes, respectively (fig. 8). Benzyl amine, 2,6-dimethyl piperazine and diethyl malonate²⁸ can be separated from sparfloxacin and eluted at 0.47, 1,38 and 6.25 minutes, respectively (fig. 9). Furthermore, impurities can be separated from the drug mixture such as ethylene diamine with N-methyl piperazine and diethyl malonate (fig. 10).

Fig.(5) HPLC Chromatogram of levofloxacin HCl (v), lomefloxacin HCl (m) , gatifloxacin (g) and sparfloxacin (s) in mixture of Leeflox^®, Lomex^®, Gatiflox^® and Spara^® tablet formulations.

Column : Chromolith^® Performance RP-18e (100 x 4.6 mm).

Mobile phase : MeOH : 0.025M KH₂PO₄ adjusted to pH 3 using

ortho phosphoric acid (20:80, v/v).

Flow rate : 4 ml/min.

pH : 3.

Fig.(6) HPLC Chromatogram of authentic levofloxacin HCl (v) in presence of N-methyl piperazine (np) and methyl chloroacetate (mc).

Column : Chromolith^® Performance RP-18e (100 x 4.6 mm).

Mobile phase : MeOH : 0.025M KH₂PO₄ adjusted to pH 3 using

ortho phosphoric acid (20:80, v/v).

Flow rate : 4 ml/min.

pH : 3.

Fig.(7) HPLC Chromatogram of authentic lomefloxacin HCl (m) in presence of 2,3,4-trifluoroaniline (f).

Column : Chromolith^® Performance RP-18e (100 x 4.6 mm).

Mobile phase : MeOH : 0.025M KH₂PO₄ adjusted to pH 3 using

ortho phosphoric acid (20:80, v/v).

Flow rate : 4 ml/min.

pH : 3.

Fig.(8) HPLC Chromatogram of authentic gatifloxacin (g) in presence of ethylene diamine (e) and 2,3,4-trifluoroaniline (f).

Column : Chromolith^® Performance RP-18e (100 x 4.6 mm).

Mobile phase : MeOH : 0.025M KH₂PO₄ adjusted to pH 3 using

ortho phosphoric acid (20:80, v/v).

Flow rate : 4 ml/min.

pH : 3.

Fig.(9) HPLC Chromatogram of authentic sparfloxacin (s) in presence of benzyl amine (ba), 2,6-dimethyl piperazine (dmp) and diethyl malonate (dem).

Column : Chromolith^® Performance RP-18e (100 x 4.6 mm).

Mobile phase : MeOH : 0.025M KH₂PO₄ adjusted to pH 3 using

ortho phosphoric acid (20:80, v/v).

Flow rate : 4 ml/min.

pH : 3.

Fig.(10) HPLC Chromatogram of authentic mixture of levofloxacin HCl (v), lomefloxacin HCl (m) , gatifloxacin (g) and sparfloxacin (s) in presence of ethylene diamine (e) with N-methyl piperazine (np) and diethyl malonate (dem).

Column : Chromolith^® Performance RP-18e (100 x 4.6 mm).

Mobile phase : MeOH : 0.025M KH₂PO₄ adjusted to pH 3 using

ortho phosphoric acid (20:80, v/v).

Flow rate : 4 ml/min.

pH : 3.

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

Parameters	Conditions
Column	Chromolith^® Performance RP-18e (100 x 4.6 mm)
Mobile phase	Isocratic binary mobile phase of MeOH : 0.025M KH₂PO₄ adjusted to pH 3 using ortho - phosphoric acid (20:80, v/v), filtered and degassed using 0.45µm membrane filter
UV detection, nm	290
Flow rate, ml/min	4
Injected volume, µl	10
Pressure, psig	2980
Temperature	Ambient

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

Parameters	Levofloxacin HCl *			Lomefloxacin HCl *			Gatifloxacin*			Sparfloxacin*
Parameters	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	1	1.003	100.31	1	1.013	101.38	1	1.002	100.25	1	1.006	100.63
	10	10.09	100.96	10	9.95	99.45	10	9.99	99.96	10	9.94	99.41
	20	19.83	99.17	20	19.90	99.51	20	20.30	101.52	20	19.90	99.52
	40	39.94	99.85	40	39.99	99.99	40	39.92	99.81	40	39.83	99.56
	60	60.13	100.22	60	60.46	100.76	60	60.22	100.36	60	60.45	100.75
	80	79.90	99.88	80	79.93	99.92	80	80.14	100.17	80	81.10	101.37
Mean			100.06			100.17			100.34			100.21
±SD			0.596			0.755			0.610			0.821
±RSD			0.595			0.754			0.607			0.820
±SE			0.243			0.308			0.250			0.335
Variance			0.355			0.570			0.456			0.675
Slope			12004			15814			15527			12976
L.D.			0.250			0.250			0.250			0.250
L.Q.			0.750			0.750			0.750			0.750
S.S.			7 x 10^-8			6 x 10^-8			5 x 10^-8			7 x 10^-8

* Average of three independent procedures.

3.2. Method Validation:

The developed methods were validated according to international conference of harmonization guidelines²⁹.

3.2.1. Linearity:

Six different concentrations of a mixture of all four drugs were prepared for linearity studies. The response was measured as peak area. The calibration curves obtained by plotting peak area against concentration showed linearity in the concentration range of 1 - 80 µg.ml^-1 for all drugs. Linear regression equation of levofloxacin HCl, lomefloxacin HCl, gatifloxacin and sparfloxacin was found to be y = 12004x + 15281, y = 15814x + 10652, y = 15527x + 22871, and y = 12976x + 13078, respectively and the regression coefficient values (r) were found to be 0.9998, 0.9997, 0.9992 and 0.9991, respectively indicating a high degree of linearity for all drugs.

3.2.2. Accuracy:

The accuracy of the method was 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).

3.2.3. precision:

Intraday precision was evaluated by calculating standard deviation (SD) of five replicate determinations using the same solution containing pure drug. The SD values revealed the high precision of the methods (values vary from 0.6 to 0.81).

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. The day - to - day SD values were in the range of 0.7 - 1.79.

3.2.4. Specificity:

The specificity studies revealed the absence of any excipent or impurity interference, since none of the peaks appeared at the same retention time of levofloxacin HCl, lomefloxacin HCl, gatifloxacin and sparfloxacin as shown in figures 6, 7, 8, 9 and 10.

3.2.5. L.D. and L.Q.:

For determining the limit of detection (L.D.) and limit of quantitation (L.Q.), the method based on signal – to - noise ratio (3:1 for L.D. and 10:1 for L.Q.) was adopted. The limit of detection for the four drugs was 0.250 µg.ml^-1 while the limit of quantitation was 0.750 µg.ml^-1 (table 2).

3.2.6. Robustness:

The robustness of the method was evaluated by making small changes in the flow rate (3.9, 4, 4.1), pH of mobile phase within a range of ± 0.2 unit of the optimized pH and mobile phase ratio keeping the other chromatographic conditions constant where the effect of the changes was studied on the percent recovery of drugs. The changes had negligible influence on the results as revealed by small SD values (≤ 1.93).

3.3. Applications:

Some Pharmaceutical formulations containing stated drugs have been successfully analyzed by the proposed method. Excipients and impurities did not show interference indicating high specificity. Results obtained were compared to those obtained by applying reference methods^{5, 6, 9, 10} where Student’s t-test and F-test were performed for comparison. Results are shown in tables 3, 4, 5 and 6 where the calculated t and F values were less than tabulated values for the four drugs which in turn indicate that there is no significant difference between proposed method and reference ones relative to precision and accuracy.

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

Parameters	Proposed method	Reference method⁽⁵⁾
N	6	6
Mean Recovery	100.05	99.95
Variance	0.436	1.450
±SD	0.660	1.596
±RSD	0.660	1.597
±SE	0.270	0.652
Student-t	0.145 (2.02)a
F-test	3.320 (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 Lomex^® tablets compared with reference method.

Parameters	Proposed method	Reference method⁽⁶⁾
N	6	6
Mean Recovery	100.15	99.97
Variance	0.635	1.480
±SD	0.797	1.659
±RSD	0.795	1.660
±SE	0.325	0.677
Student-t	0.243 (2.02)a
F-test	2.332 (5.05)b

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

Table(5). Statistical analysis of results obtained by the proposed method applied on Gatiflox^® tablets compared with reference method.

Parameters	Proposed method	Reference method⁽⁹⁾
N	6	6
Mean Recovery	100.42	99.64
Variance	0.595	1.520
±SD	0.771	1.806
±RSD	0.768	1.810
±SE	0.315	0.730
Student-t	0.982 (2.02)a
F-test	2.551 (5.05)b

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

Table(6). Statistical analysis of results obtained by the proposed method applied on Spara^® tablets compared with reference method.

Parameters	Proposed method	Reference method⁽¹⁰⁾
N	6	6
Mean Recovery	100.49	100.58
Variance	0.864	1.450
±SD	0.929	1.640
±RSD	0.924	1.635
±SE	0.380	0.670
Student-t	0.121 (2.02)a
F-test	1.672 (5.05)b

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

4. CONCLUSION:

An RP-HPLC method for rapid simultaneous estimation of levofloxacin HCl, lomefloxacin HCl, gatifloxacin and sparfloxacin within 5 minutes was developed and validated. The amounts obtained by the proposed method are between 100.05% and 100.49%, within the acceptance level of 95% to 105%. The results obtained indicate that the proposed method is rapid, accurate, selective, and reproducible. Linearity was observed over a concentration range of 1 to 80 μg.ml^-1 for all four drugs. The method has been successfully applied for the analysis of marketed tablets. 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. The main advantage of the method is the common chromatographic conditions adopted for all formulations in addition to reduced analysis time due to monolithic silica columns.

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Received on 10.07.2011 Modified on 01.08.2011

Asian J. Research Chem. 4(11): Nov., 2011; Page 1688-1694