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 (enzymecatalyzing 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 mayaccount 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 unlinkingof daughter chromosomes following replication) which is believedto be the primary target in gram-positive bacteria³.

Gatifloxacin ((±)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid ), 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) and Lomefloxacin HCl ((±)-1-ethyl-6, 8-difluoro-1,4-dihydro-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid Hydrochloride )⁴ are fluoroquinolones and antimicrobials with potent activity against a broad spectrum of bacteria.

Several HPLC methods had been developed for determination of these drugsindividually^5-13 or in combination with other drugs^14-18. Various other techniques including non aqueous titrimetry¹⁹, derivative spectrometry²⁰, capillary zone electrophoresis²¹ and linear sweep voltammetry²² have also been reported. Furthermore, Some visible spectrophotometric methods have been reported for assay of these drugs^23-29but these methods suffered from some disadvantages such as poor sensitivity, complicated experimental setup and meticulous control of experimental variables. The proposed methods were found to be accurate, very sensitive, reproducible, and consistent.

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.

· Gatifloxacin (EPCI, Egyptian Company for Pharmaceutical and Chemical Industries, S.A.E., Beni Suef, Egypt). Standard solution 500 µg.ml^-1and for molar ratio 1x10^-3M (in case of bromophenol red method) or 10 µg.ml^-1 and for molar ratio 1x10^-4M (in case of fast green FCF method) were prepared by dissolving pure drug in least amount of 0.05 M HCl then completing to 100 ml with bidistilled water.

· Levofloxacin HCl (Pharaonia Pharmaceutical Industries, Alexandria, Egypt). Standard solution 500 µg.ml^-1and for molar ratio 1x10^-3(in case of bromophenol red method) or 10 µg.ml^-1 and for molar ratio 1x10^-4M (in case of fast green FCF method) were prepared by dissolving pure drug in 100 ml bidistilled water.

· Lomefloxacin HCl (Sigma Pharmaceutical Industries, Kewesna, Egypt). Standard solution 500 µg.ml^-1and for molar ratio 1x10^-3M (in case of bromophenol red method) or 10 µg.ml^-1 and for molar ratio 1x10^-4M (in case of fast green FCF method) were prepared by dissolving pure drug in 100 ml bidistilled water.

· Acetate and borate buffer solutions of pH values 3 – 7 were prepared as in recommended methods⁽³⁰⁾.

· Bromophenol Red 0.1% (Aldrich Chemical Co. Ltd., Dorset, England) and 1x10^-3M in 20 % methanol solution as stock solution (stable for 2 weeks at least).

· Fast Green FCF 0.01% (Aldrich Chemical Co. Ltd., Dorset, England) 1x10^-4M in 10 % methanol solution as stock solution (stable for 2 weeks at least).

· Methylene chloride (El-Nasr Pharmaceutical Chemicals).

2.3. Pharmaceutical preparations:

The following available pharmaceutical preparations were analyzed

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

· 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).

2.4. Procedures:

2.4.1. General spectrophotometric procedures and construction of calibration curves using Bromophenol Red method:

Aliquot portions of gatifloxacin, levofloxacin HCl and lomefloxacin HCl of 500 µg.ml^-1 ranging from (0.2 - 1.4 ml) were transferred into a series of 125-ml separating funnels. To these, 3 ml of buffer solution (pH 3.4) and 2 ml (in case of gatifloxacin, levofloxacin HCl) or 3 ml (in case of lomefloxacin HCl) of 0.1% bromophenol red dye were added. The total volume of aqueous phase was adjusted to 10 ml with bidistilled water and 10 ml of chloroform was added twice. The contents were shaken vigorously for 5 minutes. The two phases were allowed to separate and the absorbance of the yellow colored extract was measured at 430 nm, 428 nm and 426 nm (in case of gatifloxacin, levofloxacin HCl and lomefloxacin HCl respectively) against a reagent blank similarly prepared without drug.

2.4.2. General spectrophotometric procedures and construction of calibration curves using Fast Green FCF method:

Aliquot portions of gatifloxacin, levofloxacin HCl and lomefloxacin HCl of 10 µg.ml^-1 ranging from (0.2 - 1.8 ml) were transferred into a series of 125-ml separating funnels. To these, 3 ml of buffer solution (pH 3.2) and 2 ml (in case of gatifloxacin, levofloxacin HCl) or 3 ml (in case of lomefloxacin HCl) of 0.01% fast green FCF dye were added. The total volume of aqueous phase was adjusted to10 ml with bidistilled water and 10 ml of chloroform was added twice. The contents were shaken vigorously for 5 minutes. The two phases were allowed to separate and the absorbance of the blue colored extract was measured at 631 nm against a reagent blank similarly prepared without drug.

2.4.3. Procedures for pharmaceutical preparations:

For Gatiflox^® tablets: 10 tablets were weighed and powdered. An accurately amounts of the powder equivalent to 50 mg of gatifloxacin were dissolved in least amount of 0.05 M HCl, filtered into 100-ml measuring flask and completed to volume with bidistilled water to give final concentration of 500 µg.ml^-1 (in case of bromophenol red method) or diluted to give final concentration of 10 µg.ml^-1(in case of fast green FCF method). The procedures were then completed as mentioned above under the general procedures.

For Leeflox^® and Lomex^®tablets: 10 tablets were weighed and powdered. An accurately amounts of the powder equivalent to 50 mg of levofloxacin HCl and lomefloxacin HCl were dissolved in bidistilled water, filtered into 100-ml measuring flask and completed to volume with bidistilled water to give final concentration of 500 µg.ml^-1 (in case of bromophenol red method) or diluted to give final concentration of 10 µg.ml^-1(in case of fast green FCF method). The procedures were then completed as mentioned above under the general procedures.

2.4.4. Job’s method Procedures for determination of molar ratio:

For Bromophenol Red method: gatifloxacin, levofloxacin HCl and lomefloxacin HCl and bromophenol red solutions of equimolar concentrations (1x10^-3) were prepared. Aliquots of each solution were added in different ratios so that the total volume of both was 5 ml in presence of the recommended buffer (pH 3.4). Absorbance of the yellow colored extract was measured against reagent blank at the appropriate wave length.

For Fast Green FCF method: gatifloxacin, levofloxacin HCl and lomefloxacin HCl and fast green FCF solutions of equimolar concentrations (1x10^-4) were prepared. Aliquots of each solution were added in different ratios so that the total volume of both was 5 ml in presence of the recommended buffer (pH 3.2). Absorbance of the blue colored extract was measured against reagent blank at the appropriate wave length.

3. RESULTS AND DISCUSSION:

The studied fluoroquinolones contain terminal nitrogen atom in piperazine moiety. In proper acidic medium, this nitrogen atom is protonated to give positively charged quaternary ammonium group which in turn forms an ion pair complex with negatively charged dye containing sulphonic acid group. This complex is readily extractable in dichloromethane and measured at the appropriate wavelength. The theoretical basis of this method is that the dissociation equilibrium of BA-type (which is dissociated in aqueous medium) can be shifted toward the left (association) if the ion pair is removed by extraction using a solvent immiscible with water³¹.

BA B⁺ + A^-

Where B⁺ is the protonated amine drug and A^- is the dye anion form.

3.1. Absorption spectra:

Absorption spectra of the reagents with gatifloxacin, levofloxacin HCl and lomefloxacin HCl were studied over range of 200-800 nm. Bromophenol red reacts with gatifloxacin, levofloxacin HCl, lomefloxacin HCl to yield an extractable yellow colored complex exhibiting maximum absorption at 430 nm, 428 nm and 426 nm respectively (Fig. 1). Also, fast green FCF yields a blue colored extractable complex with these drugs exhibiting maximum absorption at 631 nm (Fig. 2).

3.2. Effect of pH:

Variation in pH from 3.0 to 7.0 was investigated on the reaction of bromophenol red and fast green FCF with concerned drugs. Maximum sensitivity is obtained at pH 3.4 in case of bromophenol red and 3.2 in case of fast green FCF (Fig. 3 and 4). Different buffer volumes have been tested and optimum buffer volume was 3 ml.

Fig.(1) Absorption spectra of 0.1% bromophenol red ion-pair extractable complex with gatifloxacin (G) at 430 nm, levofloxacin HCl (V) at 428 nm and lomefloxacin HCl (M) at 426 nm in dichloromethane.

Fig.(2) Absorption spectra of 0.01% fast green FCF ion-pair extractable complex with gatifloxacin (G), levofloxacin HCl (V) and lomefloxacin HCl (M) at 631 nm in dichloromethane .

Fig.(3) Effect of pH on ion-pair complex between 0.1% bromophenol red and gatifloxacin (G), levofloxacin HCl (V) and lomefloxacin HCl (M).

Fig.(4) Effect of pH on ion-pair complex between 0.01% fast green FCF and gatifloxacin (G), levofloxacin HCl (V) and lomefloxacin HCl (M).

3.3. Effect of Reagent concentration:

Effect of reagent concentration on the intensity of absorption was studied by varying the reagent volume while other factors were held constant (Fig. 5 and 6) and optimum reagent volumes were recorded in the general procedures.

Fig.(5) Effect of dye volume of 0.1% bromophenol red with gatifloxacin (G), levofloxacin HCl (V) and lomefloxacin HCl (M).

Fig.(6) Effect of dye volume of 0.01% fast green FCF with gatifloxacin (G), levofloxacin HCl (V) and lomefloxacin HCl (M).

3.4. Effect of temperature:

Effect of temperature was studied and results showed that there is no obvious effect of temperature on these reactions as increase in temperature is not accompanied with any increase in absorbance and so optimum extraction was performed at room temperature.

3.5. Effect of solvent:

Different solvents have been tried in order to achieve maximum sensitivity and product stability such as dichloromethane, chloroform, carbon tetrachloride, ethyl acetate, petroleum ether and toluene.

Dichloromethane and chloroform showed the highest sensitivity but dichloromethane was preferred due to higher toxicity (10 times) and higher expense of chloroform³². To achieve maximum sensitivity, 10 ml of dichloromethane was added twice on two portions.

3.6. Effect of diverse ions:

The effect of diverse ions was studied using cobalt, nickel, zinc, cupper, and ferric ions and all showed sharp decrease in absorbance. This effect was suggested to be due to formation of soluble complexes of flouroquinolone drugs with these metals which are non extractable in dichloromethane.

3.7. Effect of addition sequence:

Addition sequences were studied and results revealed that the most appropriate sequence for drugs with both dyes was drug, buffer and finally dye addition.

3.8. Composition of the formed complexes:

According to stated procedures, Job’s method³⁰ revealed1:1 ratios for 1x10^-3M bromophenol red with 1x10^-3M gatifloxacin, levofloxacin HCl and lomefloxacin HCl (Fig. 7). Also, Job’s method³⁰ revealed 1:1 ratios for 1x10^-4M fast green FCF with 1x10^-4M gatifloxacin, levofloxacin HCl and lomefloxacin HCl (Fig. 8). This finding supports that the interaction of the studied drugs and the reagents takes place at only one site which was the more sterically free terminal basic nitrogen atom of piperazine moiety.

Fig.(7) Job’s method for molar ratio estimation of 1x10^-3M bromophenol red with 1x10^-3M gatifloxacin (G), levofloxacin HCl (V) and lomefloxacin HCl (M).

Table(1). Analytical parameters for the determination of gatifloxacin, levofloxacin HCl and lomefloxacin HCl using bromophenol red method.

Parameters	Bromophenol Red 0.1%
Parameters	Gatifloxacin	Levofloxacin HCl	Lomefloxacin HCl
λmax, nm	430	428	426
Volume of reagent, ml	2	2	3
pH	3.4	3.4	3.4
Beer's law limits, µg/ml	10-55	10-70	10-70
Regression equation	y=0.0144x +0.0084	y=0.0113x +0.0051	y=0.0101x +0.0051
Correlation Coefficient	0.9998	0.9994	0.9993
Molar Ratio	1:1	1:1	1:1

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). Analytical parameters for the determination of gatifloxacin, levofloxacin HCl and lomefloxacin HCl using fast green FCF method.

Parameters	Fast Green FCF 0.01%
Parameters	Gatifloxacin	Levofloxacin HCl	Lomefloxacin HCl
λmax, nm	631	631	631
Volume of reagent, ml	2	2	3
pH	3.2	3.2	3.2
Beer's law limits, µg/ml	0.2-1.8	0.2-1.8	0.2-1.8
Regression equation	y=0.3614x +0.0662	y=0.3209x +0.074	y=0.3183x +0.0505
Correlation Coefficient	0.9998	0.9997	0.9998
Molar Ratio	1:1	1:1	1:1

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

Fig.(8) Job’s method for molar ratio estimation of 1x10^-4M fast green FCF with 1x10^-4M gatifloxacin (G), levofloxacin HCl (V) and lomefloxacin HCl (M).

3.9. Method Validation:

The developed methods were validated according to international conference of harmonization guidelines⁽³³⁾.The linearity range of absorbance as a function of drug concentration (Tables 1 and 2) provides good indication about sensitivity of reagents used. 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 and 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. The SD values revealed the high precision of the methods (values vary from 0.37 to 0.85). 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 0.76 - 1.83. The robustness of the methods was evaluated by making small changes in the pH of buffer (e.g. 3.3, 3.4 and 3.5 in case of bromophenol red method) and reagent concentration 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).

According to ICH (International Conference of Harmonization) guidelines, the obtained values indicated high sensitivity of the proposed methods.

Table(3). Results of the analysis for determination of gatifloxacin, levofloxacin HCl and lomefloxacin HCl using bromophenol red method.

parameters	Bromophenol Red
	Gatifloxacin*			Levofloxacin HCl *			Lomefloxacin HCl *
	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	15	15.07	100.51	20	19.99	99.95	20	20.08	100.44
	25	24.93	99.75	30	30.07	100.26	30	30.09	100.29
	35	35.21	100.62	40	40.25	100.64	40	40.18	100.47
	45	45.42	100.95	50	49.99	99.98	50	49.79	99.58
	55	55.01	100.01	60	59.54	99.24	60	59.85	99.81
Mean			100.03			100.01			100.12
±SD			0.376			0.511			0.399
±RSD			0.376			0.511			0.398
±SE			0.168			0.231			0.178
Variance			0.142			0.262			0.161
Slope			0.0144			0.0113			0.0101
L.D.			0.242			0.312			0.348
L.Q.			0.808			1.041			1.161
S.S.			0.068			0.087			0.097
Apparent Molar absorbitivity L.Mol^-1.cm^-1			5.51x10³			4.55x10³			3.98x10³

* Average of three independent procedures.

Table(4). Results of the analysis for determination of gatifloxacin, levofloxacin HCl and lomefloxacin HCl using fast green FCF method.

parameters	Fast Green FCF
	Gatifloxacin*			Levofloxacin HCl *			Lomefloxacin HCl *
	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	0.2	0.201	100.72	0.2	0.199	99.71	0.2	0.199	99.74
	0.6	0.605	100.90	0.6	0.604	100.75	0.6	0.605	100.79
	1	0.993	99.28	1	1.000	100.00	1	0.988	98.80
	1.4	1.399	99.97	1.4	1.399	99.94	1.4	1.412	100.87
	1.8	1.809	100.50	1.8	1.794	99.71	1.8	1.795	99.75
Mean			100.27			100.09			99.99
±SD			0.657			0.449			0.851
±RSD			0.655			0.448			0.851
±SE			0.293			0.201			0.383
Variance			0.432			0.201			0.735
Slope			0.3614			0.3209			0.3183
L.D.			0.012			0.013			0.013
L.Q.			0.041			0.045			0.045
S.S.			0.002			0.002			0.002
Apparent Molar absorbitivity L.Mol^-1.cm^-1			1.81x10⁵			1.79x10⁵			1.58x10⁵

* Average of three independent procedures.

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

Parameters	Bromophenol Red method	Fast Green FCF method	Reference method⁽²⁵⁾
N	5	5	5
Mean Recovery	100.37	100.36	100.01
Variance	0.235	0.522	1.210
±SD	0.485	0.722	1.353
±RSD	0.483	0.719	1.353
±SE	0.217	0.320	0.605
Student-t	0.456 (2.57)a	0.515 (2.57)a
F-test	5.140 (6.256)b	2.311 (6.256)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 methods applied on Leeflox^® tablets compared with reference method.

Parameters	Bromophenol Red method	Fast Green FCF method	Reference method⁽²⁷⁾
N	5	5	5
Mean Recovery	99.80	99.99	100.21
Variance	0.293	0.340	1.301
±SD	0.541	0.580	1.441
±RSD	0.542	0.580	1.438
±SE	0.242	0.261	0.644
Student-t	0.610 (2.57)a	0.314 (2.57)a
F-test	4.430 (6.256)b	3.823 (6.256)b

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

3.10. Applications:

Some Pharmaceutical formulations containing stated drugs have been successfully analyzed by the proposed methods. Excipients did not show interference indicating high specificity. Results obtained were compared to those obtained by applying reference methods^25,27,28 where Student’s t-test and F-test were performed for comparison. Results are shown in tables 5, 6 and 7 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 precision and accuracy.

Table (7). Statistical analysis of results obtained by the proposed methods applied on Lomex^® tablets compared with reference method.

Parameters	Bromophenol Red method	Fast Green FCF method	Reference method⁽²⁸⁾
N	5	5	5
Mean Recovery	100.01	100.18	99.84
Variance	0.240	0.826	1.051
±SD	0.489	0.909	1.226
±RSD	0.489	0.907	1.229
±SE	0.218	0.401	0.550
Student-t	0.282 (2.57)a	0.493 (2.57)a
F-test	4.382 (6.256)b	1.275 (6.256)b

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

4. CONCLUSION:

Unlike GC and HPLC techniques, spectrophotometry is simple and inexpensive. The proposed methods require only dyes as reagents which are cheaper and readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. Morever, both methods are simple, fast, accurate, adequately sensitive and free from interference by common additives and excipients which make it as choice for routine quality control analysis. The amounts obtained by the proposed methods are between 99.80% and 100.37%, within the acceptance level of 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 tablets.

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Received on 10.07.2011 Accepted on 17.08.2011

Asian J. Pharm. Tech. 1(4): Oct. - Dec. 2011; Page 130-136