Spectrofluorimetric Determination of Certain Fluoroquinolones Through Charge Transfer Complex Formation

A highly sensitive spectrofluorimetric method was developed for the first time, for the analysis of ten fluoroquinolones (FQs) antibacterials, namely amifloxacin (AMI), ciprofloxacin (CIP), difloxacin (DIF), enoxacin (ENO), enrofloxacin (ENR), lomefloxacin (LOM), levofloxacin (LEV), norfloxacin (NOR), ofloxacin (OFL) and pefloxacin (PEF) in their pharmaceutical dosage forms or in biological fluids through charge transfer (CT) complex formation with bromanil (BRO). The BRO was found to react with these drugs to produce stable complexes and the fluorescence intensity of the complexes was greatly enhanced. The formation of such complexes was also confirmed by ultravioletvisible measurements. The different experimental parameters that affect the fluorescence intensity were carefully studied. At the optimum reaction conditions, the drug-BRO complexes showed excitation maxima ranging from 275 to 290 nm and emission maxima ranging from 450 to 470 nm. Rectilinear calibration graphs were obtained in the concentration range 0.02 to 3.1 μg.mL for the studied drugs. The method has been successfully applied to determine their pharmaceutical dosage forms with good precision and accuracy compared to official and reported methods as revealed by tand F-tests. They are also applied for the determination of studied drugs in human urine samples.


INTRODUCTION
Flurorquinolones (FQs) are a class of important synthetic antibiotics, which are active against both Gram (+) and Gram (-) bacteria through inhibition of their DNA gyrase [1] , also they have some activity against myobacteria, mycoplasmas and rickettsias.

Apparatus:
Perkin Elmer TM luminescence spectrometer model LS 45 (Perkin Elmer instruments, USA) connected to an IBM computer loaded with the FLwinlab TM application software 4.00.02 version was used. All the measurements took place in a standard 10 mm path-length quartz cell, thermo stated at 25.0±0.5 o C, with 2.5 nm bandwidth for the emission and excitation monochromators.
Spectronic TM Genesys TM , ultraviolet-visible spectrophotometer, (Milton Roy Co., USA) with matched 1cm quartz cells was used, connected to an IBM computer loaded with the Winspec TM application software.
General procedure: A suitable amount of drug solution was pipetted into a 10-mL volumetric flask, 1.0 mL of BRO solution was added and the solution was diluted to volume with acetone and mixed thoroughly. The solution was thermostated at 25 Analysis of pharmaceutical dosage forms Analysis of tablets: An accurately weighed amount, equivalent to 10 mg of each drug from composite of 20 powdered tablets, was transferred into a 100 mLcalibrated flask and diluted to the mark with the appropriate solvent, sonicated for 20 min and filtered off to obtain solutions of 100 µg.mL -1 . Further dilutions were made to obtain sample solution and the general procedure was proceedd earlier.

Analysis of ampoules:
A volume equivalent to 10 mg of each drug was transferred into 100 mL-calibrated flask and diluted to the mark with the appropriate solvent to obtain solution of 100 µg.mL -1 . Further dilutions were made to obtain sample solution and the general procedure was proceeded earlier.

Analysis of drops:
One milliliter of the drops was transferred into a 100 mL-calibrated flask and diluted to the mark with the appropriate solvent to obtain a solution of 30 µg. mL -1 .Further dilutions were made to obtain sample solution and the general procedure was proceeded as under earlier.
Analysis of human urine: Dilute urine samples of a healthy subject who has taken, orally, FQs tablets at specific times, in 5-mL sample solution were transferred into a separating funnel and shaken well for 3 min. Then 5 mL of 0.2 mol l -1 phosphate buffer solution (pH 7.0) was added and the mixture shaken and extracted with 3 x 10 mL of dichloromethane chloroform (1:1 v/v) mixture. The organic layer was filtered over anhydrous sodium sulfate. The extract was dried under nitrogen gas at room temperature and residue dissolved in least amount of acetone, transferred into a 10 mL-volumetric flask and procedure was proceeded as under earlier.

RESULTS AND DISCUSSION
Excitation spectra and emission spectra: Solution of the studied drugs have native fluorescence, however in presence of BRO, the fluorescence intensity increases substantially (Fig. 1). Indicated CT complexes formation between the investigated drugs and BRO, these complexations probably occurs through the lone pair of electron donated by the N atom in piperazinyl of FQs (n-donor) to BRO (π-acceptor).

Effect of reaction temperature:
The effect of temperature on the formed CT complexes was studied in the range of 10-60 o C. All the formed complexes were stable up to 40 o C, at temperatures higher than 40 o C, the relative fluorescence intensity decreases due to dissociation of the complexes. Similarly, dependence of the fluorescence intensity on reaction temperature be neglected in the range of 10-40 o C, thus the determination of studied drugs were carried out at 25±0.5 o C. It was further found that it takes 30 min for formation of the complexes which were stable for at least 24 h.

Effect of BRO concentration:
The influence of CT reagent concentration was studied in the range 4 x 10 -5 mol l -1 -4 x 10 -3 mol l -1 . The relative fluorescence intensity increased with increasing BRO concentration up to 4 x 10 -4 mol l -1 but leveled off at higher concentrations. Experiments indicated that 1.0 mL BRO solution is enough for each drug.
Effect of solvent: Fluorescence spectral characteristics of AMI, CIP, DIF, ENO, ENR, LOM, LEV, NOR, OFL and PEF in different solvents are compared. The studied solvents involved water, methanol, ethanol, isopropanol, acetone, acetoniltrile and chloroform.  Experimental results indicated that acetone gave the maximum and stable fluorescence emission for studied drugs.

Effect of CT reagent:
The influence of the CT reagent on the relative fluorescence intensity of all the formed CT complexes with FQs was studied at their respective maxima using BRO, TCNQ, TCNE, CL and DDQ as model electron acceptors. The results show that BRO is most sensitive CT reagent for the studied drugs. In general, the order of decreasing sensitivity is BRO > TCNQ > TCNE > DDQ > Cl (Fig. 2). Figure 3 and Table 1 show maximum absorbance of ten FQs in acetone ranging from 276 to 315 nm. When BRO solution was added to studied drugs solution, the studied drugs solution with BRO cause an immediate change in the absorption spectrum with new characteristic bands at 470-480 nm. The appearance of a new band in the visible region of the spectrum was evidence for the formation of a CT complex between the studied components and BRO.

Mechanism of reaction:
BRO is an π-acceptor, AMI, CIP, DIF, ENO, ENR, LOM, LEV, NOR, OFL and PEF are nitrogenous compounds. So CT complexes can be formed with these drugs. Molar ratio of the reactants in the CT complex was determined by Job ' s method of continuous variation [29] and Yoe and Jones method of mole ratio [30] and it was found to be 1:1 for all the studied drugs with BRO. This ratio may be due to the presence of the bromine atom acting as an electron      drawing group in the molecule of FQs. The benzene ring has lower electron density, but nitrogen atom in 4 of piperazinyl has more electron density and is less sterically hindered. So n-π CT complexes were formed ( Table 2).
Analytical parameters: Under the experimental conditions described, standard calibration curves of CT complexes for AMI, CIP, DIF, ENO, ENR, LOM, LEV, NOR, OFL and PEF with BRO were constructed by plotting fluorescence intensity versus concentration, the linear regression equations, analytical and statistical parameters for each drug are listed in Table 3. The correlation coefficients ranged from 0.9995 to 0.9999, indicating good linearity. The small value of variance confirmed the small degree of scattering of the experimental data points around the regression line. Precision of the proposed method was determined for each drug in its concentration range, by 10 measurements carried out on different days within 1 week (Table 4). Target concentrations corresponded to middle values in each range. Table 3 gives a R.S.D. (within-day and between-day) of solutions of 0.01, 0.10 and 1.00 µg mL -1 determined using the proposed procedure.

Analysis of pharmaceutical formulations:
The proposed method was applied to the determination of the studied drugs in their pharmaceutical formulations. Five replicate determinations were made. Satisfactory results were obtained for studied drugs (Table 5). Moreover, to check the validity of the proposed methods, the standard addition technique was applied by adding AMI, CIP, DIF, ENO, ENR, LOM, LEV, NOR, OFL and PEF to the previously analyzed pharmaceutical formulations. The recovery of each drug was calculated by comparing the concentration obtained from the (spiked) mixtures with those of the pure drugs. Table 6 shows the results of analysis of the commercial pharmaceutical formulations and the recovery study (standard addition technique) of studied drugs. Comparison of the results obtained by the proposed method with those obtained by reference method [16] indicated that the accuracy and precision are satisfactory. The obtained high-intensity fluorescence bands and the very low reagent background make this procedure suitable for the routine quality control analysis of the investigated drugs with minimum interference.

Analysis of human urine:
The proposed method was applied to determine CIP as a representative example of the studied drugs in human urine samples from healthy volunteers who received a single oral dose of 500 mg CIP. The urine samples of individuals were collected at 6, 12, 24 and 36 hr after oral administration of CIP tablets. In this case, the high performance liquid chromatography (HPLC) method proposed by Wong et al. was used as a reference method [31] . The results obtained summarized in Table 6, show that both methods (spectrofluorimetric and chromatographic) yield values within the same range when compared statistically.
The accuracy was assessed by investigating the recovery of each of the studied drugs at four concentration levels covering the specified range (five replicates of each concentration). The results showed average percentage recoveries to be 98.1±0.4 with standard deviations less than 2.0 for human urine, indicating both good accuracy and precision ( Table 6).
The increase in sensitivity obtained with the proposed method, compared with other methods is very substantial. Comparison with other method for the determination of studied drugs, described in the literature (Table 7), showed an improvement of about one order of magnitude against HPLC methods. The HPLC methods generally require complex and expensive equipment, provision for use and disposal of solvents, labor-intensive sample preparation procedures and personnel skilled in chromatographic techniques. For spectrofluorimetry through charge transfer reaction, its main advantage over HPLC methods [32][33][34] is its rapidity; method possesses good analytical selectivity, higher capacity against blank interference and can improve the limit of detection when compared with spectrophotomeric methods [16] .

CONCLUSION
The results obtained from the present study indicate that the complex formed between the studied FQs and BRO can be employed in the spectrofluotimetric assay of AMI, CIP, DIF, ENO, ENR, LOM, LEV, NOR, OFL and PEF in dosage forms and human urine. The proposed method is suitable for the routine quality control of the drug alone, in different pharmaceutical formulations and in human urine without fear of interference caused by the excipients expected to be present in pharmaceutical formulations or components of human urine.