Colourimetric and atomic absorption spectrometric determination of some f luoroquinolone derivatives

Three simple, accurate, sensitive and selective procedures for the determination of ten fluoroquinolones (amifloxacin, ciprofloxacin hydrochloride, difloxacin hydrochloride, enoxacin, enrofloxacin, lomefloxacin hydrochloride, lovefloxacin, norfloxacin, ofloxacin and pefloxacin mesylate) were described. Procedures I and II are based on the formation of ion-pair complexes between the drugs and ammonium reineckate reagent in an acidic medium at 25 eOc and the formed precipitates are quantitatively determined either colourimetrically (procedure I) or by atomic absorption spectrometrically (procedure 11). Procedure I is based on dissolving the formed precipitate with acetone, the volume was completed quantitatively and the absorbance of the solution was measured at 527 nm against pure solvent blank. The formed precipitates on the atomic absorption spectrometric procedure (procedure II) are quantitatively determined either directly or indirectly through the chromium precipitate formed or the residual un-reacted chromium in the filtrate at 358.6 nm and the optimum conditions for precipitation have been carefully studied. Procedure Ill is based on the reaction of the studied drugs with 2,2-diphenylI-picrylhydrazyl reagent (DPPH). The latter is employed to abstract a hydrogen atom from the drugs thereby promoting a process of radical coupling. This results in a reduction of the violet color of DPPH with the formation of the yellow colored 2,2-diphenyl-1 -picrylhydrazine (DPPH2). Tel.: +20-10-142-8104; fax: +20-86-369-075, E-mail address: h-salem-eg @ yahoo.com (H. Salem).

either colourimetrically (procedure I) or by atomic absorption spectrometrically (procedure 11).Procedure I is based on dissolving the formed precipitate with acetone, the volume was completed quantitatively and the absorbance of the solution was measured at 527 nm against pure solvent blank.The formed precipitates on the atomic absorption spectrometric procedure (procedure II) are quantitatively determined either directly or indirectly through the chromium precipitate formed or the residual un-reacted chromium in the filtrate at 358.6 nm and the optimum conditions for precipitation have been carefully studied.
Procedure Ill is based on the reaction of the studied drugs with 2,2-diphenyl-I-picrylhydrazyl reagent (DPPH).The latter is employed to abstract a hydrogen atom from the drugs thereby promoting a process of radical coupling.This results in a reduction of the violet color of DPPH with the formation of the yellow colored 2,2-diphenyl-1 -picrylhydrazine (DPPH2).

Introduction
Nalidixic acid was used primarily in the treatment of urinary tract infections but the problems of development of bacterial resistance or superinfection with inherently resistant species severely limited its use [I].
Recently, synthesis of the new quinolones with 6-fluoro and 7-piperazinyl groups resulted in enhanced activity against a wide range of gram negative and gram positive bacteria [2].Many analytical procedures have been adopted for the determination of this class of compounds.The USP-XXIII and National Formulary-XVIII-(1 995) specifies a non-aqueous titration method for norfloxacin bulk drug and an HPLC one for its dosage forms and for ciprofloxacin bulk drug and dosage forms [3], other methods are based on spectrophotometric [4-91, fluorometric [ I 0-1 21 and polarographic [ I 31 techniques.Many chromatographic methods [14-201 have been described.A number of visual spectrophotometric methods for the determination of individual compounds such as norfloxacin [lo, 121, ciprofloxacin [ I 5, 16 1,   levofloxacin [g, 11, 13, 151 and enoxacin [8] are reported in the literature, few have been reported for other compounds.It is noteworthy to mention that, there is no general method for their determination as a large chemical group.
Reineckts salt is ammonium tetrathiocyanotodiamminochromate (Ill) monohydrate in which it can be used for quantitative determination of many pharmaceutical compounds applying gravimetric [21], titrimetric [22] and spectrophotometric [23] procedures.
2,2-Diphenyl-1-Picrylhydrazyl (DPPH) is an intense, violet-colored, stable, free radical which reacts as a chromogenic reagent [24,25] by extracting a hydrogen atom to form the yellow colored diphenylpicrylhydrazine (DPPH*).This decrease in the intensity of the violet color is used as a measure of the quantity of the tested drugs.The radical DPPH was chosen for the present work because it does not dimerize and the problem of cage effect does not arise.In addition, it is highly colored and its concentration at any time can be estimated by its absorption in the visible range.
The purpose of the present work is to describe the development of two simple and accurate spectrophotometric procedures, as well as a selective and sensitive atomic absorption spectrometric procedure, for the analysis of the titled antibiotics in the pure form as well as in pharmaceutical preparations.

Pharmaceutical preparations
The following available commercial preparations were analyzed:

Ammonium reineckate
Ammonium reineckate (Prolabo, Paris, France) were of purity not less than 99.9%.3x1 M stock ammonium reineckate solutions were freshly prepared by dissolving in re-distilled de-ionized water.

DPPH
2,2-Diphenyl-1 -picrylhydrazyl (Sigma, St. Louis, Mo, USA).A stock solution of 1.5 mg ml-' was prepared by dissolving 0.15 g of DPPH in methanol and then diluted to 100 ml with the same solvent.Ten milliliter of this solution were diluted to 100 ml to give 0.15 mg ml-' (working DPPH solution).The stock and working solutions were kept in a refrigerator and protected from light.The solution was found to be stable for at least one week at 4OC.

Procedure I
In a 10 ml volumetric flask, 2 ml volume of sample drug solution, 0.5 ml of hydrochloric acid was added, 2.5 ml of fresh saturated reinecke1s salt solution was added with agitation for 5 min.and complete to the volume with re-distilled de-ionized water.The formed precipitate was filtered through a sintered glass funnel (G4) after one hour and washed thrice with 5 ml ice water.Then the precipitate was dried in a vacuum desiccator.The formed precipitate in the crucible was then dissolved with acetone into a 25 ml volumetric flask together with the successive washings of the funnel and filtration device.The volume was completed quantitatively with acetone to the appropriated volume and the absorbance of the solution was measured at 527 nm against pure solvent as a blank.

Direct procedure
The above (drug-reineckate) precipitates were collected on a G4 sintered glass crucible and washed with five 2 ml portions of ice water.The drugs reineckate precipitates were dissolved in 25 ml acetone.The solution was nebulized in an air-acetylene flame of AAS measurement of chromium at 358.6 nm.The absorbance was compared with a calibration graph prepared from the pure drug-reineckate solid complex under identical conditions.

Indirect procedure
The filtrate and washings from the direct procedure 11 (2.5.2.1) were collected in 100 ml volumetric flask and completed to volume with acetone.
The resulting solution (2 ml) was diluted to 25 ml with acetone.
A blank (omitting addition of drugs) was prepared and absorbance was measured at the above flaming conditions.Chromium concentration was calculated from a calibration curve.

Procedure 111
Transfer 1 ml of the investigated drug to a 10 ml volumetric flask, add 2 ml of DPPH solution.Mix well for enrofloxacin at 25OC, heat in water bath at

Tablets
The contents of twenty tablets of the drug were thoroughly ground.A quantity equivalent to 50 mg drug was accurately weight into a 100 ml volumetric flask, completed to volume with the appropriated solvent, filtered, and the procedures were completed as under procedures 1-111.

Vials
An accurately measured weight of vials equivalent to 50 mg of drug was dissolved in the appropriated solvent, (by shaking for 5 minutes), filtered if necessary.The solution was completed to 100 ml with re-distilled de-ionized water for procedures I and I 1 and with methanol for procedure Ill.The procedures were completed as under procedures 1-111.

Drops
Aliquot of ~i p r o " otic drops equivalent to 50 mg ciprofloxacin hydrochloride was transferred into a 100 ml volumetric flask and completed to volume with the appropriated solvent.The procedures were completed as under procedures 1-111.

Results and Discussion
Mixing each aqueous solution of amifloxacin, ciprofloxacin hydrochloride, difloxacin hydrochloride, enoxacin, enrofloxacin, lomefloxacin hydrochloride, lovefloxacin, norfloxacin, ofloxacin and pefloxacin mesylate with ammonium reineckate in acidic medium at 25+2'~ resulted in formation of red precipitate.It is based on the formation of ion-pair complexes between the drugs and ammonium reineckate (procedures I and 11).
On the first colourimetric procedure, the absorption spectrum of the reaction products was measured at 527 nm.
On the atomic absorption spectrometric procedure, acidic solutions of the drugs gave purple coagulated precipitates with ammonium reineckate.
These precipitates form the basis of the micro-quantitative determinations of the cited fluoroquinolones.The chromium ion content could be determined either directly in the precipitate or indirectly in the filtrate.The different variables that affect the determinations of all the studied fluoroquinolones with ammonium reineckate reagent (procedures I and 11) and DPPH reagent (procedure Ill) were studied and optimized.

Reineckate methods
The cited fluoroquinolones reineckate salts were prepared starting with the same concentration of the drug and the ammonium reineckate while varying the type and amounts of the acid.The absorbencies of the final salt solutions in the appropriate solvent were taken as a measure of better precipitation.Some 0.5 ml hydrochloric acid per 10 ml of the final solution mixture was the most suitable amount for complete precipitation of the cited fluoroquinolones.

DPPH method
The effect of pH on color reduction was studied for the investigated drugs.The results revealed that the reaction was independent on pH.
Because of this independence of the reaction on pH, further investigations were not carried out to establish whether the constituents or pH range of the buffer solutions have not any effect on the interaction of DPPH with the investigated drugs.

Ammonium reineckate concentration
The general procedures (I and II) were applied using different concentrations of the reagent while the cited fluoroquinolones and acid concentrations were constant.The absorbencies of the final salt solutions in the appropriate solvent were taken as a measure of better precipitation.
A total of 2.5 ml of 2% ammonium reineckate was sufficient for complete precipitation of fluoroquinolones-hydrochloric acid in the final solution.

DPPH concentration
DPPH is added in excess to that required to react with drugs to be analyzed.By measuring the excess reagent, the consumed DPPH would correspond to the amount of the drug.
The concentration of the reagent that gives the highest absorption value within the participle sensitivity range of absorbance was found to be 0.15 mg ml".Two milliliters of this solution per 10 ml of the reaction mixture was used.

Reineckate methods
A series containing equal concentrations of the fluoroquinolones was analyzed using the corresponding standard procedure, but filtering the precipitate after various time intervals.The absorbance of the final fluoroquinolones-reineckate solution in the appropriate solvent were taken as a measure for the best precipitation time.
One hour was found to be sufficient for complete precipitation of fluoroquinolones-reineckate on standing at 25 OC.
The effect of temperature on the formation of the coloured precipitate was investigated.The experiments were carried out at room temperature (25+2OC).

DPPH method
The reaction time was determined for the interaction of each studied

Solubility and stability of the precipitated reineckates
Trials to find out the best solvent to dissolve fluoroquinolonesreineckate precipitate were performed using distilled water, acetone, dioxan, methanol and ethanol.Then the stability of the produced colour in each solvent was examined periodically at different time intervals over 24 hours.
The colour of fluoroquinolones-reineckate acetone solution was stable for at least 24 hours.

Reineckate methods
The washing liquid of choice was ice water and acetone was the most suitable solvent.

DPPH method
The effect of dilution of the reaction product by different solvents namely, methanol, ethanol, n-propanol, isopropanol, n-butanol, acetone was studied.The results indicated that all solvents had no effect on the position of maximum absorption while the reactivity (AA value) was affected.Methanol was found to be the most suitable solvent.

I. Reineckate methods
Standard curves were constructed by plotting the observed absorbencies readings versus the concentrations of fluoroquinolones in pg ml-' of the final solution of the experiments.Conformance to Beer's law was evident.Plots showed good linearity with high correlation coefficients (Table 3).

DPPH method
Under the optimum parameters, ~e e r ' s law was obeyed for all studied drugs in the range shown in Table 3.The regression analysis of AA value versus concentration were done for all the studied drugs according to the linear regression equation summarized in Table 3.

Validation of the proposed procedures
Accuracy, precision, specificity, detection limit, quantitation limit and linearity range were tested (Tables 3 and 4).
Statistical analysis of the results obtained by the proposed procedures (I,II and Ill) compared with those of the official methods [27] for ciprofloxacin hydrochloride, norfloxacin and ofloxacin are given in table 3, where the remaining fluoroquinolones are not official, in which they are compared with reported methods [28-301.The calculated t and F values do not exceed the tabulated ones, revealing equal precision and accuracy (Table 4).

Reineckate methods
For the atomic absorption spectrometric method, the Job's method of continuous variation [38] indicated a molar ratio of 1:2 drug to reineckate (Figure 2).a: intercept; b: slope; r: correlation coefficient; LOD: limit of detection; LOQ; limit of quantitation; procedure IIa: direct atomic absorption spectroscopic method; procedure 11~: indirect atomic absorption spectroscopic method.Three and six determinations were used for the reported and the reference methods, respectively.The tabulated values of t and F at 95% confidence limit are t=2.23 and F=5.79.

DPPH method
The Job's method of continuous variation [31] indicated a molar ratio of 1:l drug to DPPH reagent (Figure 3).Such results obtained have been indicating that one abstractable hydrogen is present in the drug molecule.

DPPH method
The ultraviolet-visible spectra of the assay solution of one of the tested drugs, DPPH and DPPH2 are shown in Figure 1.The reaction is assumed to proceed via abstraction of hydrogen atoms from the drugs by DPPH.This is accompanied by the change of violet color of DPPH to give the yellow colored DPPH2 and the corresponding free radical of the drug (Scheme 1).

Interferences
Before dealing with the analysis of the pharmaceutical preparations, the effect of common additives, adjuvants and excepients on the proposed methods were experimentally studied.The results obtained, revealed that hydroxypropyl methylcellulose, microcrystalline cellulose, sodium stearyl fumarate, titanium dioxide, talc, iron oxide red, iron oxide yellow, glucose, lactose, magnesium stearate and starch do not interfere.

Application
The applicability of the methods to various dosage forms were checked by analyzing synthetic mixtures containing the cited fluoroquinolones.
The results of the analysis of pharmaceutical preparations by the suggested procedures (I, II and Ill) are comparable to the official and reported methods and show good correlation and reveal good applicability without interference (Table 5).
The proposed methods are non-specific with regard to differentiation between the different fluoroquinolones.This shortcoming does not affect the utility of the method in routine analysis and content uniformity determination of these drugs as they singly prescribed.The enhanced sensitivity allows for additional advantage of handling without fear of increasing errors.
As conclusion, the developed methods are simpler, faster, more sensitive and accurate than the official titrimetric and chromatographic methods.In addition, the atomic absorption spectrometric method is selective and suitable for routine quality control.
Table 5 Statistical analysis of the results obtained using the proposed procedures and reference methods for analysis of fluoroquinolones in their pharmaceutical preparations.Three and six determinations were used for the reported and the reference methods, respectively.The tabulated values of t and F at 95% confidence limit are t=2.23 and F=5.79.
60 O C for 20 minutes for amifloxacin, difloxacin, enoxacin, lomefloxacin hydrochloride and pefloxacin mesylate and at 70 O C for 15 minutes for ciprof loxacin hydrochloride, lovefloxacin, norfloxacin and of loxacin.Cool and complete to the mark with methanol.Measure the absorbencies of the sample and a reagent blank against methanol at h=520 nm.Calculate AA, i.e. the difference between the absorbance values of the blank and sample, which corresponds to the drug concentration.
The ultraviolet-visible spectra of the assay solution of one of the tested drugs, DPPH and DPPH* (procedure Ill) are shown in Figure 1.The reaction is assumed to proceed via abstraction of hydrogen atoms from the drugs by DPPH [26].This is accompanied by the change of violet color of DPPH to give the yellow colored DPPH*.Measure the absorbencies of the sample and a reagent blank against methanol at h=520 nm.
drug with DPPH solution by following the color reduction at ambient temperature (25 + 2 OC), 30, 40, 50, 60 and 70 OC.The optimum temperature and reaction time were recorded in table 2. The AA reached a constant level, at once and remained constant for at least 180 minutes.
Figure 2. Job's plot of continuous variation between ofloxacin and reineck's salt.

Figure 3 .
Figure 3. Job's plot of continuous variation between ofloxacin and DPPH reagent