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.

Sparfloxacin (5-Amino-1-cyclopropyl-7-(cis-3,5-dimethyl-1-piperazinyl) -6,8- difluoro-1,4 dihydro-4-oxo-3-quinolinecarboxylic acid) and Gatifloxacin ((±)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid) are fluoroquinolones and antimicrobials with potent activity against a broad spectrum of bacteria.

Metronidazole {2-methyl-5-nitroimidazole-1-ethanol} and Tinidazole {1-(2-ethyl-sulphonyl ethyl) -2-methyl-5-nitroimidazole} are used as antiamoebic, antiprotozoal and antibacterial agents⁴.

Some HPLC methods had been developed for determination of these drugs individually^5-9 or in combination with other drugs^10-13 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 sparfloxacin, gatifloxacin, metronidazole and tinidazole. 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).

· Sparfloxacin (Global Napi), Gatifloxacin (EPCI), Metronidazole (Sanovi Aventis) and Tinidazole (Medical Union of Pharmaceuticals). 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, 2,3,4-trifluoroaniline, 2,6-dimethyl piperazine, Diethyl malonate and 2-methyl imidazole were supported from Merck, Germany.

2.3. Pharmaceutical preparations:

The following available pharmaceutical preparations were analyzed

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

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

· Flagyl^® tablets labeled to contain 500 mg metronidazole per tablet. Batch No. 10E70 (Sanovi Aventis, Egypt).

· Protozole^® tablets labeled to contain 500 mg tinidazole per tablet. Batch No. 90133 (MUP, Egypt).

2.4. Procedures:

2.4.1. Preparation of calibration curves:

Appropriate mixed dilutions of the standard stock solutions of sparfloxacin, gatifloxacin, metronidazole and tinidazole 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 formulationwere weighed and powdered. 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 Soga⁽¹⁴⁾. 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 separation¹⁵ (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 porosity¹⁶.

3.1. Optimization of Chromatographic Conditions:

All chromatographic conditions are illustrated in table 1. Spectroscopic analysis of the drugs showed that sparfloxacin, gatifloxacin, metronidazole and tinidazole have maximum UV absorbance (λ_max) at 291 nm, 290 nm, 300 nm and 298 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. Furthermore, It was observed that 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 ml/min. Under these conditions, sparfloxacin, gatifloxacin, metronidazole and tinidazole were eluted at 4.3, 3, 1.8, and 1.2 minutes respectively with a run time of 10 minutes. A typical chromatogram for simultaneous estimation of these drugs obtained by using the aforementioned mobile phase is illustrated in figures 3 (authentic mixture) and 4 (tablet formulations).

Fig.(3) HPLC Chromatogram of authentic mixture of sparfloxacin (s), gatifloxacin (g) , metronidazole (r) and tinidazole (t).

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 methods.

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 methods.

parameters	Sparfloxacin*			Gatifloxacin*			Metronidazole *			Tinidazole*
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	1.006	100.63	1	1.002	100.25	1	1.018	101.87	1	0.996	99.61
	10	9.94	99.41	10	9.99	99.96	10	10.04	100.42	10	10.13	101.34
	20	19.90	99.52	20	20.30	101.52	20	20.07	100.35	20	20.03	100.15
	40	39.83	99.56	40	39.92	99.81	40	39.59	98.97	40	39.64	99.09
	60	60.45	100.75	60	60.22	100.36	60	59.78	99.63	60	60.33	100.55
	80	81.10	101.37	80	80.14	100.17	80	80.34	100.43	80	79.91	99.88
Mean			100.21			100.34			100.28			100.10
±SD			0.821			0.610			0.970			0.781
±RSD			0.820			0.607			0.967			0.780
±SE			0.335			0.250			0.396			0.319
Variance			0.675			0.456			0.940			0.610
Slope			12976			15527			8529.4			6282.1
L.D.			0.250			0.250			0.300			0.300
L.Q.			0.750			0.750			0.900			0.900
S.S.			7 x 10^-8			5 x 10^-8			1 x 10^-7			2 x 10^-7

* Average of three independent procedures.

Fig.(4) HPLC Chromatogram of drugs in mixture of Spara^®, Gatiflox^®, Flagyl^® and Protozole^® 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.(5) 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.

Also, chromatographic conditions were appropriate for separation of each drug from its manufacturing impurities. Benzyl amine, 2,6-dimethyl piperazine and diethyl malonate¹⁷ can be separated from sparfloxacin and eluted at 0.47, 1,38 and 6.25 minutes, respectively (fig. 5). Ethylene diamine and 2,3,4-trifluoroaniline¹⁸ can be separated from gatifloxacin and eluted at 0.53 and 1.19 minutes, respectively (fig. 6). 2-methyl imidazole⁽¹⁹⁾ can be separated from tinidazole and metronidazole and eluted at 0.86 minute (fig. 7).

Fig.(6) 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.

3.2. Method Validation:

The developed methods were validated according to international conference of harmonization guidelines²⁰.

Fig.(7) HPLC Chromatogram of authentic tinidazole (t) and metronidazole (r) in presence of 2-methyl imidazole (mi).

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.

3.2.1. Linearity:

Six different concentrations of a mixture of all drugs in both methods 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 sparfloxacin, gatifloxacin, metronidazole and tinidazole was found to be y = 12976x + 13078, y = 15527x + 22871, y = 8529.4x – 567.18 and y = 6282.1x + 273.23 respectively and the regression coefficient values (r) were found to be 0.9991, 0.9992, 0.9999 and 0.9995 respectively indicating a high degree of linearity for all drugs.

3.2.2. Accuracy:

The accuracy of the methods 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 0.78 to 0.96). 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 0.98 - 1.9.

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 sparfloxacin, gatifloxacin, metronidazole and tinidazole as shown in figures 5, 6 and 7.

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. & 10:1 for L.Q.) was adopted. The limit of detection for both sparfloxacin and gatifloxacin was 0.250 µg.ml^-1and for both metronidazole and tinidazole was 0.300 µg.ml^-1 while the limit of quantitation for both sparfloxacin and gatifloxacin was 0.750 µg.ml^-1and for both metronidazole and tinidazole was 0.900 µg.ml^-1 (table 2).

3.2.6. Robustness:

The robustness of the methods 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).

3.2.7. Applications:

Some Pharmaceutical formulations containing stated drugs in combination and in pure form have been successfully analyzed by the proposed methods. Excipients and impurities did not show interference indicating high specificity. Results obtained were compared to those obtained by applying reference methods^{7,
9, 11, 13} 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 norfloxacin, tinidazole and metronidazole which in turn indicate that there is no significant difference between proposed methods and reference ones relative to precision and accuracy.

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

Parameters	Proposed method	Reference method⁽⁹⁾
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.

Table(4). Statistical analysis of results obtained by the proposed method applied on Gatiflox^® tablets compared with reference method.

Parameters	Proposed method	Reference method⁽⁷⁾
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(5). Statistical analysis of results obtained by the proposed method applied on Flagyl^® tablets compared with reference method.

Parameters	Proposed method	Reference method⁽¹³⁾
N	6	6
Mean Recovery	99.98	100.71
Variance	0.981	1.550
±SD	0.991	1.243
±RSD	0.990	1.235
±SE	0.404	0.507
Student-t	1.130 (2.02)a
F-test	1.581 (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 Protozole^® tablets compared with reference method.

Parameters	Proposed method	Reference method⁽¹¹⁾
N	6	6
Mean Recovery	99.55	100.65
Variance	0.637	1.445
±SD	0.798	1.331
±RSD	0.802	1.330
±SE	0.325	0.545
Student-t	1.722 (2.02)a
F-test	2.263 (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 sparfloxacin, gatifloxacin, metronidazole and tinidazole within 5 minutes was developed and validated. The amounts obtained by the proposed method are between 99.55% 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^-1for 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 Accepted on 14.08.2011

Asian J. Pharm. Res. 1(4): Oct. - Dec. 2011; Page 119-125