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.

Norfloxacin is 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid and is antimicrobial with potent activity against a broad spectrum of bacteria. Tinidazole is 1-(2-ethyl-sulphonyl ethyl)-2-methyl-5-nitroimidazole and is used as antiamoebic, antiprotozoal and antibacterial agents⁴. Literature survey revealed that a few number of methods have been reported for estimation of norfloxacin and tinidazole individually^5-10 or in combination form^11-12but No HPLC method for simultaneous estimation of these two drugs using monolithic silica columns has been reported till date. In the present study, an attempt has been made to develop a method for rapid simultaneous estimation of norfloxacin 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).

· Norfloxacin and Tinidazole (Wockhardt). 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, Ethylene diamine, 2-methyl imidazole, Piperazine, and Methyl chloroacetate were supported from Merck, Germany.

2.3. Pharmaceutical preparations:

The following available pharmaceutical preparations was analyzed: Conaz^®tablets labeled to contain 400 mg norfloxacin and 600 mg tinidazole per tablet. Batch No. 9160055 (Wockhardt, Egypt).

2.4. Procedures:

2.4.1. Preparation of calibration curves:

Appropriate mixed dilutions of the standard stock solutions of norfloxacin 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 both 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 both drugs.

2.4.2. Sample preparation:

10 tablets of Conaz^® formulation were weighed and powdered. An accurately amounts of the powder equivalent to 40 mg of norfloxacin 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⁽¹³⁾. 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 separation¹⁴ (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 porosity¹⁵.

3.1. Optimization of Chromatographic Conditions:

All chromatographic conditions are illustrated in table 1. Spectroscopic analysis of the drugs showed that norfloxacin and tinidazole have maximum UV absorbance (λ_max) at 280 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. 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 4.0 ml/min. Under these conditions, tinidazole and norfloxacin in pharmaceutical formulation can be separated and eluted at 1.2 and 1.6 minutes respectively. A typical chromatogram for simultaneous estimation of both drugs obtained by using the aforementioned mobile phase in authentic mixture and in tablet formulation is illustrated figures 3 and 4, respectively.

Fig.(3) HPLC Chromatogram of authentic mixture of tinidazole (t) and norfloxacin (n) at pH 3.

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	Norfloxacin*			Tinidazole*
Parameters	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	1	0.996	99.65	1	0.996	99.61
	10	10.03	100.3	10	10.13	101.34
	20	20.1	100.51	20	20.03	100.15
	40	40.68	101.71	40	39.64	99.09
	60	60.11	100.19	60	60.33	100.55
	80	79.59	99.48	80	79.91	99.88
Mean			100.30			100.10
±SD			0.790			0.780
±RSD			0.787			0.780
±SE			0.322			0.319
Variance			0.624			0.610
Slope			8238.8			6282.1
L.D.			0.250			0.300
L.Q.			0.750			0.900
S.S.			10 x 10^-8			2 x 10^-7

* Average of three independent procedures.

Fig.(4) HPLC Chromatogram of tinidazole (t) and norfloxacin (n) in Conaz^® 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 norfloxacin (n) in presence of ethylene diamine (e), piperazine (p) 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.

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 method (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 norfloxacin and tinidazole as shown in figures 5, 6 and 7.

Fig.(6) HPLC Chromatogram of authentic tinidazole (t) 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.

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

Parameters	Proposed method		Reference method⁽¹¹⁾
Parameters	(Norfloxacin)	(Tinidazole)	(Norfloxacin)	(Tinidazole)
N	6	6	6	6
Mean Recovery	100.40	99.55	100.03	100.65
Variance	0.730	0.637	1.510	1.445
±SD	0.850	0.798	1.681	1.331
±RSD	0.848	0.802	1.680	1.330
±SE	0.348	0.325	0.680	0.545
Student-t	0.481 (2.02)a	1.722 (2.02)a
F-test	2.061 (5.05)b	2.263 (5.05)b

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

Fig.(7) HPLC Chromatogram of authentic mixture of tinidazole (t) and norfloxacin (n) in presence of ethylene diamine (e) with 2-methyl imidazole (mi) 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.

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 norfloxacin was 0.250 µg.ml^-1and for tinidazole was 0.300 µg.ml^-1 while the limit of quantitation for norfloxacin was 0.750 µg.ml^-1and for 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:

Conaz^® Pharmaceutical formulation containing combination of norfloxacin and tinidazole had 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 method⁽¹¹⁾ where Student’s t-test and F-test were performed for comparison. Results are shown in table 3 where the calculated t and F values were less than tabulated values for norfloxacin and tinidazole which in turn indicate that there is no significant difference between proposed method and reference one relative to precision and accuracy.

4. CONCLUSION:

An RP-HPLC method for rapid simultaneous estimation of norfloxacin and tinidazole within 2 minutes was developed and validated. The amounts obtained by the proposed method are between 99.55% and 100.4%, 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 both drugs. The method has been successfully applied for the analysis of marketed tablet Conaz^®. The main advantage of the method is the reduced analysis time due to monolithic silica columns. This analytical method is also adequate and useful for the estimation of norfloxacin and tinidazole in tablet for quality control laboratories, where low cost and fast analysis are essential.

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