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 compounds⁽¹⁾.

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 classes⁽²⁾.

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 bacteria⁽³⁾.

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-quinolinecarboxylicacid Hydrochloride) and Sparfloxacin (5-Amino-1-cyclopropyl-7-(cis-3,5-dimethyl-1-piperazinyl)-6,8-difluoro-1,4-dihydro- 4-oxo-3-quinolinecarboxylicacid)⁽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^(14-17). Various other techniques including non aqueous titrimetry⁽¹⁸⁾, derivative spectrometry⁽¹⁹⁾, capillary zone electrophoresis⁽²⁰⁾ and differential pulse voltammetry⁽²¹⁾ have also been reported. Furthermore, Some visible spectrophotometric methods have been reported for assay of these drugs⁽^22-28)but 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.

2.2. Materials and reagents:

· All solvents and reagents were of analytical grade and double distilled water was used throughout the work.

· Levofloxacin HCl (Pharaonia Pharmaceutical Industries, Alexandria, Egypt). Standard solution 10 µg.ml^{-1 was} prepared by dissolving pure drug in 100 ml bidistilled water.

· Lomefloxacin HCl (Sigma Pharmaceutical Industries, Kewesna, Egypt). Standard solution 10 µg.ml^-1
was prepared by dissolving pure drug in 100 ml bidistilled water.

· Sparfloxacin (Global Napi Pharmaceuticals, 6 october, Egypt). Standard solution 10 µg.ml^-1was prepared by dissolving pure drug in least amount of DMF then completing to 100 ml with bidistilled water.

· 5 M HCl (El-Nasr Chemicals, Egypt) was prepared by diluting 225 ml of concentrated HCl (36%) to 500 ml.

· Methylene Blue 60 µg/ml (Universal Fine Chemicals, India) was dissolved in 20 ml methanol then completed to 100 ml with bidistilled water (stable for 2 weeks at least).

· Methyl Orange 60 µg/ml (Universal Fine Chemicals, India) was dissolved in 20 ml methanol then completed to 100 ml with bidistilled water (stable for 2 weeks at least).

· Thymol Blue 100 µg/ml (Aldrich Chemical Co. Ltd., Dorset, England) was dissolved in 80 ml ethanol then completed to 100 ml with bidistilled water (stable for 2 weeks at least).

· Bromate / Bromide stock solution was prepared by dissolving 0.1 gm of potassium bromate (Winlab, England) and 1.0 gm of potassium bromide (Winlab, England) in 100 ml bidistilled water (stable for 10 days at least). Working solution was freshly prepared daily by diluting 2.5 ml of stock solution to 100 ml with bidistilled water (25 µg/ml in case of methylene blue), 1.25 ml of stock solution (12.5 µg/ml in case of methyl orange) or 2.7 ml of stock solution (27 µg/ml in case of thymol blue).

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).

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

2.4. Procedures:

2.4.1. General spectrophotometric procedures and construction of calibration curves using Methylene Blue method:

To 1 ml bromate - bromide working solution in 10 - ml volumetric flasks, add 0.1 - 2 ml (in case of levofloxacin HCl and lomefloxacin HCl) or 0.1 - 1.4 ml (in case of sparfloxacin) drug solution then acidify using 0.6 ml 5 M HCl, close flasks and stand for 10 minutes, add 1 ml dye working solution then stand for another 10 minutes and complete to mark with bidistilled water then measure absorbance against reagent blank similarly prepared without drug at 678 nm.

2.4.2. General spectrophotometric procedures and construction of calibration curves using Methyl Orange method:

To 1 ml bromate - bromide working solution in 10 - ml volumetric flasks, add 0.2 - 2 ml (in case of levofloxacin HCl and lomefloxacin HCl) or 0.1 - 1.8 ml (in case of sparfloxacin) drug solution then acidify using 0.6 ml 5 M HCl, close flasks and stand for 10 minutes, add 1 ml dye working solution then stand for 2 minutes and complete to mark with bidistilled water then measure absorbance against reagent blank similarly prepared without drug at 510 nm.

2.4.3. General spectrophotometric procedures and construction of calibration curves using Thymol Blue method:

To 1 ml bromate - bromide working solution in 10 - ml volumetric flasks, add 0.5 – 5.5 ml (in case of levofloxacin HCl and lomefloxacin HCl) or 1 - 5.5 ml (in case of sparfloxacin) drug solution then acidify using 0.6 ml 5 M HCl, close flasks and stand for 10 minutes, add 1 ml dye working solution then stand for 2 minutes and complete to mark with bidistilled water then measure absorbance against reagent blank similarly prepared without drug at 545 nm.

2.4.4. Procedures for pharmaceutical preparations:

For Leeflox^® and Lomex^®tablets: 10 tablets were weighed and powdered. An accurately amounts of the powder equivalent to 100 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 1000 µg.ml^-1 then diluted to give final concentration of 10 µg.ml^-1. The procedures were then completed as mentioned above under the general procedures.

For Spara^® tablets: 10 tablets were weighed and powdered. An accurately amounts of the powder equivalent to 100 mg of sparfloxacin were dissolved in in least amount of DMF, filtered into 100-ml measuring flask and completed to volume with bidistilled water to give final concentration of 1000 µg.ml^-1 then diluted to give final concentration of 10 µg.ml^-1. The procedures were then completed as mentioned above under the general procedures.

3. RESULTS AND DISCUSSION:

The proposed spectrophotometric methods are indirect and are based on the determination of the residual bromine (insitu generated) after allowing the reaction between each drug and a measured amount of bromine to be complete. The surplus bromine was determined by reacting it with a fixed amount of either methylene blue, methyl orange or thymol blue dye. The methods rely on the bleaching action of bromine on the dyes due to oxidative destruction of these dyes as shown in figure 1 (in case of methylene blue). Levofloxacin HCl, lomefloxacin HCl or sparfloxacin when added in increasing amounts to a fixed amount of insitu generated bromine, consume the latter proportionately with a concomitant fall in the concentaration of bromine. When a fixed amount of dye is added to the decreasing amounts of bromine, a concomitant increase in the concentration of dye results. Consequently, a proportional increase in the absorbance at the respective λ_max is observed with increasing concentration of each drug.

The insitu generation of bromine is carried out using a mixture of potassium bromate and potassium bromide in presence of 5 M HCl according to the following equation:

5Br^-+ BrO₃^-+ 6H⁺3Br₂+ 3H₂O

3.1. Absorption spectra:

Absorption spectra for determination of levofloxacin HCl, lomefloxacin HCl and sparfloxacin were studied over range of 200 - 800 nm. After oxidation of both drugs and portions of dyes with bromine, residual unoxidized methylene blue, methyl orange and thymol blue are absorbed at 678, 510 and 545 nm respectively (Fig. 2, 3 and 4).

3.2. Effect of Acidity:

Different acids were tested as a medium for bromine generation including sulphuric acid, hydrochloric acid, nitric acid and phosphoric acid. Hydrochloric acid produced the most precise and accurate results. Therefore, 5 M HCl was used throughout experiments and it was found that 0.6 ml of 5 M HCl (accurately measured) is the appropriate acid volume and increasing HCl volume results in a rapid decrease in absorption (Fig. 5, 6 and 7).

3.3. Effect of bromate - bromide volume:

Bromate - bromide volume was studied by varying the reagent volume while other factors were held constant. It was found that 1 ml of bromine is sufficient for its bleaching action using these stated concentrations (25, 12.5, 27 µg/ml for methylene blue, methyl orange and thymol blue, respectively) (Fig. 8, 9 and 10).

3.4. Effect of time:

Time required to brominate and oxidize the drug before addition of dye and time required to irreversibly oxidize dye after its addition was studied. The bromination reaction was found to be complete in 10 minutes for levofloxacin HCl, lomefloxacin HCl and sparfloxacin while contact times up to 25 minutes had been examined and no further bromination was detected (Fig. 11, 12 and 13). A contact time of 10 minutes (in case of methylene blue) (Fig. 14) or 2 minutes (in case of methyl orange and thymol blue) (Fig. 15 and 16) was necessary for the bleaching of the dye colour by the residual bromine and the colour of the three dyes remains stable for at least two hours after mixing with the reaction mixture.

3.5. Method validation:

The developed methods were validated according to international conference of harmonization guidelines⁽³⁰⁾.The linearity range of absorbance as a function of drug concentration (Tables 1, 2 and 3) 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 4, 5 and 6). 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 0.29 to 0.79). 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 0.63 - 1.72. The robustness of the methods was evaluated by making small changes in the volume of acid (0.55, 0.6 and 0.65) and contact time 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.

3.6. 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⁽^{22, 26, 27)} where Student’s t-test and F-test were performed for comparison. Results are shown in tables 7, 8 and 9 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.

Fig.(2) Absorption spectra of 60µg/ml methylene blue in case of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) after bromine oxidation at 678 nm.

Fig.(3) Absorption spectra of 60µg/ml methyl orange in case of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) after bromine oxidation at 510 nm.

Fig.(4) Absorption spectra of 100 µg/ml thymol blue in case of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) after bromine oxidation at 545 nm.

Fig.(5) Effect of volume of 5M HCL in case of methylene blue (60µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 678 nm.

Fig.(6) Effect of volume of 5M HCL in case of methyl orange (60µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 510 nm.

Fig.(7) Effect of volume of 5M HCL in case of thymol blue (100µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 545 nm.

Fig.(8) Effect of volume of Bromate-Bromide mixture (25µg/ml) in case of methylene blue (60µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 678 nm.

Fig.(9) Effect of volume of Bromate-Bromide mixture (12.5µg/ml) in case of methyl orange (60µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 510 nm.

Fig.(10) Effect of volume of Bromate-Bromide mixture (27µg/ml) in case of thymol blue (100µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 545 nm.

Fig.(11) Effect of time before addition of methylene blue (60µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 678 nm.

Fig.(12) Effect of time before addition of methyl orange (60µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 510 nm.

Fig.(13) Effect of time before addition of thymol blue (100µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 545 nm.

Fig.(14) Effect of time after addition of methylene blue (60µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 678 nm.

Fig.(15) Effect of time after addition of methyl orange (60µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 510 nm.

Fig.(16) Effect of time after addition of thymol blue (100µg/ml) in presence of levofloxacin HCl (V), lomefloxacin HCl (M) and sparfloxacin (S) at 545 nm.

Table(2). Analytical parameters for the determination of levofloxacin HCl, lomefloxacin HCl and sparfloxacin using methyl orange method.

Parameters	Methyl Orange (60µg/ml)
Parameters	Levofloxacin HCl	Lomefloxacin HCl	Sparfloxacin
λmax, nm	510	510	510
Volume of dye, ml	1	1	1
Volume of 5M HCL, ml	0.6	0.6	0.6
Volume of Bromate - Bromide mixture (12.5µg/ml) , ml	1	1	1
Time before dye addition, min	10	10	10
Time after dye addition, min	2	2	2
Beer's law limits, µg/ml	0.1-1.0	0.1-1.0	0.1-1.8
Regression equation	y=0.6135x+0.1757	y=0.5938x+0.1065	y=0.3933x+0.0934
Correlation Coefficient	0.9995	0.9996	0.9994

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). Analytical parameters for the determination of levofloxacin HCl, lomefloxacin HCl and sparfloxacin using thymol blue method.

Parameters	Thymol Blue (100µg/ml)
Parameters	Levofloxacin HCl	Lomefloxacin HCl	Sparfloxacin
λmax, nm	545	545	545
Volume of dye, ml	1	1	1
Volume of 5M HCL, ml	0.6	0.6	0.6
Volume of Bromate - Bromide mixture (27µg/ml) , ml	1	1	1
Time before dye addition, min	10	10	10
Time after dye addition, min	2	2	2
Beer's law limits, µg/ml	0.25-2.75	0.25-2.75	1.0-5.5
Regression equation	y=0.2234x+0.0643	y=0.2210x+0.0361	y=0.1181x+0.0293
Correlation Coefficient	0.9997	0.9994	0.9999

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(4). Results of the analysis for determination of levofloxacin HCl, lomefloxacin HCl and sparfloxacin using methylene blue method.

Parameters	Methylene Blue
	Levofloxacin HCl *			Lomefloxacin HCl *			Sparfloxacin*
	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	0.1	0.104	100.76	0.1	0.103	100.24	0.1	0.104	100.69
	0.2	0.199	99.82	0.2	0.202	101.05	0.2	0.197	98.89
	0.4	0.398	99.56	0.4	0.397	99.28	0.4	0.402	100.51
	0.6	0.603	100.54	0.6	0.601	100.13	0.6	0.599	99.89
	0.8	0.795	99.45	0.8	0.797	99.69	0.8	0.792	99.01
Mean			100.06			100.12			99.76
±SD			0.664			0.682			0.778
±RSD			0.663			0.681			0.781
±SE			0.296			0.305			0.348
Variance			0.441			0.464			0.607
Slope			0.7804			0.5784			0.4385
L.D.			0.008			0. 011			0.014
L.Q.			0.027			0.036			0.049
S.S.			0.0005			0.0006			0.0007
Apparent Molar absorbitivity L.Mol^-1.cm^-1			7.25x10⁵			6.54x10⁵			4.99x10⁵

* Average of three independent procedures.

Table (5). Results of the analysis for determination of levofloxacin HCl, lomefloxacin HCl and sparfloxacin using methyl orange method.

Parameters	Methyl Orange
	Levofloxacin HCl *			Lomefloxacin HCl *			Sparfloxacin*
	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	0.2	0.198	99.11	0.2	0.201	100.62	0.2	0. 201	100.05
	0.4	0.397	99.23	0.4	0.395	98.73	0.6	0.594	99.03
	0.6	0.602	100.35	0.6	0.597	99.50	1.0	1.008	100.86
	0.8	0.808	101.12	0.8	0.809	101.14	1.4	1.402	100.19
	1.0	1.001	100.11	1.0	0.999	99.95	1.8	1.796	99.82
Mean			99.98			99.99			99.99
±SD			0.835			0.945			0.661
±RSD			0.835			0.945			0.661
±SE			0.373			0.423			0.296
Variance			0.698			0.894			0.438
Slope			0.6135			0.5938			0.3933
L.D.			0. 004			0.004			0.007
L.Q.			0.015			0.015			0.023
S.S.			0.001			0.001			0.002
Apparent Molar absorbitivity L.Mol^-1.cm^-1			3.93x10⁵			3.32x10⁵			2.18x10⁵

* Average of three independent procedures.

Table (6). Results of the analysis for determination of levofloxacin HCl, lomefloxacin HCl and sparfloxacin using thymol blue method.

parameters	Thymol Blue
	Levofloxacin HCl *			Lomefloxacin HCl *			Sparfloxacin*
	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	0.5	0.495	99.11	0.5	0.502	100.36	1.0	1.011	101.01
	1.0	1.001	100.13	1.0	1.013	101.31	2.0	1.982	99.11
	1.5	1.502	100.18	1.5	1.511	100.72	3.0	3.021	100.73
	2.0	1.995	99.75	2.0	1.995	99.75	4.0	4.011	100.27
	2.5	2.532	101.28	2.5	2.492	99.71	5.0	5.000	100.00
Mean			100.09			100.37			100.22
±SD			0.795			0.676			0.736
±RSD			0.794			0.674			0.735
±SE			0.355			0.302			0.329
Variance			0.632			0.457			0.543
Slope			0.2234			0.2210			0.1181
L.D.			0.013			0.014			0.026
L.Q.			0.045			0.047			0.087
S.S.			0.003			0.004			0.008
Apparent Molar absorbitivity L.Mol^-1.cm^-1			1.09x10⁵			1.01x10⁵			5.12x10⁴

* Average of three independent procedures.

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

Parameters	Methylene Blue method	Methyl Orange method	Thymol Blue method	Reference method⁽²⁶⁾
N	5	5	5	5
Mean Recovery	100.16	100.02	99.94	100.21
Variance	0.439	0.521	0.597	1.301
±SD	0.662	0.722	0.770	1.441
±RSD	0.661	0.721	0.770	1.438
±SE	0.296	0.322	0.345	0.644
Student-t	0.071 (2.57)a	0.264 (2.57)a	0.375 (2.57)a
F-test	2.960 (6.256)b	2.491 (6.256)b	2.173 (6.256)b

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

Table (8). Statistical analysis of results obtained by the proposed methods applied on Lomex^® tablets compared with reference method.

Parameters	Methylene Blue method	Methyl Orange method	Thymol Blue method	Reference method⁽²⁷⁾
N	5	5	5	5
Mean Recovery	100.21	100.28	100.11	99.84
Variance	0.632	0.628	0.485	1.051
±SD	0.795	0.793	0.696	1.226
±RSD	0.793	0.790	0.695	1.229
±SE	0.355	0.354	0.310	0.550
Student-t	0.562 (2.57)a	0.673 (2.57)a	0.432 (2.57)a
F-test	1.661 (6.256)b	1.670 (6.256)b	2.160 (6.256)b

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

Table (9). Statistical analysis of results obtained by the proposed methods applied on Spara^® tablets compared with reference method.

Parameters	Methylene Blue method	Methyl Orange method	Thymol Blue method	Reference method⁽²²⁾
N	5	5	5	5
Mean Recovery	100.01	100.16	100.54	99.75
Variance	0.661	0.838	0.780	1.450
±SD	0.810	0.915	0.883	1.620
±RSD	0.810	0.914	0.879	1.618
±SE	0.363	0.410	0.395	0.724
Student-t	0.320 (2.57)a	0.491 (2.57)a	0.963 (2.57)a
F-test	2.192 (6.256)b	1.730 (6.256)b	1.852 (6.256)b

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

Parameters	Methylene Blue (60µg/ml)
Parameters	Levofloxacin HCl	Lomefloxacin HCl	Sparfloxacin
λmax, nm	678	678	678
Volume of dye, ml	1	1	1
Volume of 5M HCL, ml	0.6	0.6	0.6
Volume of Bromate - Bromide mixture (25µg/ml) , ml	1	1	1
Time before dye addition, min	10	10	10
Time after dye addition, min	10	10	10
Beer's law limits, µg/ml	0.05-1.0	0.05-1.0	0.1-1.4
Regression equation	y=0.7804x+0.1792	y=0.5784x+0.1755	y=0.4385x+0.1379
Correlation Coefficient	0.9996	0.9991	0.9998