INTRODUCTION:

Moxifloxacin is a fourth generation 8-methoxy fluoroquinolone derivative [1-cyclopropyl-6-fluoro-1,4-dihydro-8- methoxy-7-{(4aS,7aS–octa-hydro-6H-pyrrolol (3,4b) pyridin-6-yl)}-4-oxo-3-quinoline carboxylic acid, monohydrochloride] with extended-spectrum and improved activity against Grampositive bacteria (including staphylococci, streptococci, enterococci), anaerobes and atypical bacteria^1-2. The bactericidal activity of moxifloxacin is mediated by the inhibition of DNA gyrase (topoisomerase II) and topoisomerase IV, essential enzymes involved in bacterial DNA replication, transcription, repair and recombination³. Moxifloxacin is prescribed for the bacterial infections of the respiratory tract including sinusitis, community acquired pneumonia and acute exacerbations of chronic bronchitis⁴. Due to clinical advantages of moxifloxacin, there has been increase in number of moxifloxacin formulations in market in recent past. A survey of literature has revealed, numerous publications describing the determination of moxifloxacin using, HPLCS^5-11, UV^11-15, voltametric^16-17flourometry¹⁸and Differential Pulse Polarographic method.¹⁹

Thorough survey of literature revealed that, although quantification of drugs based on ion-pair complexation is simple, sensitive and accurate, the studies involving any dye towards quantification of moxifoxacin is has not been reported yet. In this communication quantitative determination of moxifloxacin using bromothymolol blue (BTB), bmophenol blue (BPB) and bromocresol green (BCG) is described and the methods have been validated in the lines of ICH.

MATERIALS AND METHODS:

Moxifloxacin hydrochloride is procured from Hetero labs limited, Hyderabad as a gift sample. The dyestuffs viz., BTB, BPB and BCG (AR grade) supplied by SD Fine Chemicals Ltd. Mumbai, are used without any further purification. The dyestuffs were used as 0.025% solutions in doubly distilled water. Sodium acetate-hydrochloric acid buffers²⁰ of pH 2.5, 2.8 and 3.5 were prepared by mixing 50ml of 1.0M sodium acetate solution with 50.50, 49.50 or 46.25 ml, respectively, of 1.0 M HCl solution and diluted to 250 ml with doubly distilled water. The pH of each solution was adjusted to an appropriate value with the aid of a pH meter. Chlorofom (HPLC grade) supplied by SD Fine Chemicals Ltd. Mumbai is used throughout the work. Stock solutions were prepared for all the dyes and drugs (25mg/100ml).

The spectra (Fig. 1) of ion-pair complexes have been recorded on SHIMADZU 140 double beam spectrophotometer, Thermo Nicolet 1000 and also on ELICO 159 UV-Visible single beam spectrophotometer using quartz cells of 10 mm path length. An Elico model Li-120 pH meter was used for pH measurement.

Fig. 1. Absorption spectra of Moxifloxacin hydrochloride-dye complex extracted into 10 ml chloroform: (a) drug = 25 mg ml^-1 + 5 ml of 0.025% BTB + 5 ml of pH 2.8 buffer; (b) drug = 25 mg ml^-1 + 5 ml of 0.025% BPB + 5 ml of pH 2.5 buffer; (c) drug = 25 mg ml^-1 + 5 ml of 0.025% BCG + 5 ml of pH 3.5 buffer

Fig. 2 Calibration graphs for BCG, BPB and BTB ion pair complexes.

Calibration curve:

Different aliquots of drug solution were transferred into 125 ml separating funnel. To this 5 ml of buffer (pH 2.5 2.8 and 3.5), 5 ml of dye were added and total volume was made up to 20 ml with water. 10 ml of chloroform was added and the contents were shaken for 5 min. The two layers were allowed to separate for 5 min. The organic layer was separated and absorbance of yellow colored solution which is stable at least for 3 hrs is measured at 415,416 and 417 nm against blank similarly prepared. The same procedure of analysis is followed either for assay of pure drug or for dosage form. The calibration graphs (Fig. 2) are linear over the concentration ranges are within the permissible range. The optical characteristics and statistical data for the regression equation of the proposed methods are presented in (Table 1).

Procedure for the assay of pure drug:

Four different solutions of pure drug in the range of calibration curve were selected and the recovery experiments were performed. The recoveries and their relative standard deviations are tabulated in (Table 2).

Procedure for the assay of dosage forms:

One tablet of moxicip-400 mg are powdered and dissolved in doubly distilled water and stirred thoroughly, filtered through a Whatman No. 42 filter paper. This solution was transferred into 100 ml standard volumetric flask and diluted with doubly distilled water as required. Different solutions of drug in the range of calibration curve were chosen and the assay was estimated using the calibration curve. The results of the recovery experiments are tabulated in (Table 3).

RESULTS AND DISCUSSION:

Moxifloxacin hydrochloride forms ion-pair complexes in acidic buffer with dyestuffs such as bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol green (BCG) and these complexes are quantitatively extracted into chloroform. Ion-pair complexes of drug with BTB, BPB and BCG absorbed maximally at 415, 416 and 417 nm respectively. The reagent blank under similar conditions showed no absorption.

In order to establish molar ratio between Moxifloxacin hydrochloride and dyestuffs used, the Job’s method of continuous variation²¹ has been applied. In this method, solutions of drug and dyestuff with identical molar concentrations [8 x 10^-5M] were mixed in varying volume ratios in such a way that the total volume of each mixture was the same. The absorbance of each solution was measured and plotted against the mole fraction of the drug, [drug]/ [drug] + [dyestuff] (Fig. 3). This measurement showed that 1:1 complex was formed with each dyestuff. The formation constants^22,23 were also estimated and found to be 1.404x10⁶, 3.163x 10⁵ and 2.80x10⁵K M^-1for complexes with BTB, BPB and BCG respectively.

Fig. 3 Continuous-variations study of drug-dye systems: [Drug] = [Dye] = 8x10^-5M

TABLE -1: OPTICAL CHARACTERISTICS AND STATISTICAL FOR THE REGRESSION EQUATION OF THE PROPOSED METHODS

Parameters

Extraction methods with

BTB

BPB

BCG

λ_max (nm)

Beer’s law limit (μg ml^-1)

Molar absorptivity (L mol^-1 cm^-1)

Formation constant, K, M^-1

Sandell sensitivity (μg cm^-2)

Slope (specific absorptivity), b

Intercept (a)

Correlation coefficient (r)

Standard deviation of intercepts (% n=6)

Limit of detection, μgml^-1

Limit of quantification, μgml^-1

Regression equation

415

2.5 - 25

27497

1.403x 10⁶

0.02272

0.044

0.008

0.996

0.007937

0.5952

1.7856

Y= 0.044C +0.008

416

2.5 - 25

24423

3.163x 10⁵

0.02564

0.039

0.026

0.996

0.006807

0.5813

1.7439

Y= 0.039C +0.026

417

2.5-25

32002

2.804x 10⁵

0.02173

0.046

0.007

0.996

0.002121

0.152

0.45

Y= 0.046C +0.007

^aWith respect to Y=bc+a, where C is the concentration (μg ml^-1) and Y is absorbance

^bSix replicate samples.

TABLE – 2: APPLICATION OF PROPOSED METHODS FOR THE ANALYSIS OF MOXIFLOXACIN HYDROCHLORIDE IN PURE FORM

Taken (μg ml^-1)	Proposed methods						Reference method^[18]
	Found (μg ml^-1)			Recovery (%)			Recovery (%)
	BTB	BPB	BCG	BTB	BPB	BCG	Recovery (%)
4 8 12 16 RSD (%)	4.021 7.914 11.914 16.064	3.978 8.0473 12.02242 15.9261	4.021 8.013 11.914 16.064	98.913 99.444 100.186 99.719 0.53433	99.45 100.591 100.2016 99.5381 0.54612	100.523 99.466 100.357 100.403 0.484499	100.2167 0.570
Mean±SD t-test F-test				99.5655±0.532 1.9926 1.9584	99.945±0.5458 0.8168 2.0609	100.18±0.4858 0.1534 1.63434	100.2167±.5688

TABLE – 3: APPLICATION OF PROPOSED METHODS FOR THE ANALYSIS OF PIOGLITAZONE HYDROCHLORIDE IN PHARMACEUTICALS FORM

Taken (μg ml^-1)	Proposed methods						Reference method^[18]
	Found (μg ml^-1)			Recovery (%)			Recovery (%)
	BTB	BPB	BCG	BTB	BPB	BCG	Recovery (%)
moxicip 400mg/tab 4 8 12 16 RSD (%)	4.021 7.914 11.914 16.064	3.978 8.0473 12.02242 15.9261	4.021 8.013 11.914 16.064	100.5434 100.555 100.186 100.28 .1852	100.549 100.295 100.202 100.461 0.1564	100.525 100.533 100.178 100.269 0.1796	100.678 0.1690
Mean±SD t-test F-test				100.3911±0.1866 2.6224 2.718	100.3768±0.1570 3.1244 1.837	100.376±0.18028 2.838 2.531	100.678±0.1702

Moxifloxacin hydrochloride contains piperidine nitrogen which is protonated in acid medium, while sulphonic acid group is present in BTB, BPB and BCG, that is the only group undergoing dissociation in the pH range 1-5. The colour of such dyes is due to the opening of lactoid ring and subsequent formation of quinoid group. It is supposed that the two tautomers are present in equilibrium but due to strong acidic nature of the sulphonic acid group, the quinoid body must predominate. Finally the protonated Moxifloxacin hydrochloride forms ion-pairs with the dyestuffs which are quantitatively extracted into chloroform. The possible reaction mechanisms are proposed and given in (Scheme 1).

Moxifloxacin- bromo cresol green complex

Moxifloxacin- bromo thymolol blue complex

Moxifloxacin- bromo phenol blue complex

Scheme 1 Drug-dye complex

The influence of pH on the ion-pair formation of Moxifloxacin hydrochloride with various dyestuffs has been studied using sodium acetate-hydrochloric acid buffer. The results are shown in (Fig. 4). It is evident that absorbance of complexes with BTB, BPB and BCG was found to be constant within the pH ranges 2.0-3.0, 2.0-3.0 and 3.0-4.0 respectively. Thus, all the absorbance measurements were made at pH 2.8, 2.5 and 3.5 with BTB, BPB and BCG respectively.

Fig.4

The effect of dyestuff concentrations was also studied by adding different volumes of dyestuff to a constant amount of Moxifloxacin hydrochloride (12.5 µg ml^-1). It is apparent from (Fig. 5). That the maximum absorbance, in each case, was found with 3.0 ml of dyestuff, beyond which absorbance was constant. Thus, 5 ml of each dyestuff was used for ion-pair formation throughout the experiment.

Fig. 5 Influence of the volume of 0.025% Dye [Drug] = [12.5µg ml^-1]

A systematic study of the effect of foreign species present along with Moxifloxacin hydrochloride on the determination of Moxifloxacin hydrochloride at 12.5 µg ml^-1 levels was undertaken. This study was carried out by following the proposed procedures for a 10 ml sample system, by adding a known amount of foreign species to a Moxifloxacin hydrochloride solution of 12.5 µg ml^-1. (Table 4) summarizes the results obtained. However, the drug content from the powdered capsules was extracted into chloroform, which completely removes any interference by the common excipients found in formulations.

TABLE – 4: INTERFERENCE STUDY

S. No.

Excipients

Tolerance limit (μg ml^-1)

Microcrystalline cellulose

Starch

Lactose

Magnesium stearate

Colloidal silicon dioxide

Titanium dioxide

165

130

Validation of the proposed method:

All the three proposed methods have been validated in terms of guideline proposed by ICH²⁴ viz. selectivity, specificity, accuracy, precision, limits of calibration curve, LOD, LOQ, robustness, ruggedness and regression equation. The student t-test and variance F-test have been performed in comparison with a reference method. (Table 1) summarizes the values for Beer’s law limits, molar absorptivity, regression equation, correlation coefficients, relative standard deviation and recoveries. To test the reproducibility of the proposed methods, six replicate determinations of 4µg ml^-1of Moxifloxacin hydrochloride were made. The coefficient of variation was found to be less than 1.2% for all the procedures.

The proposed methods have been successfully applied to the determination of Moxifloxacin hydrochloride in pharmaceutical preparations. The performance order of the proposed methods is BCG>BTB>BPB. The results obtained and shown in (Table 2 and Table 3) were compared to those obtained by a reference method¹⁴ by means of t-test at 95% confidence level. In all cases, the average results obtained by proposed methods and reference method were statistically identical, as the difference between the average values had no significance at 95% confidence level.

The proposed methods are simple, sensitive and reproducible and can be used for routine analysis of Moxifloxacin hydrochloride in pure form and in formulation.

CONCLUSION:

Moxifloxacin hydrochloride formed ion pair complexes with acidic dyes with 1:1 composition and extractable in to chloroform for assay of the drug. The method is validated and applied to pharmaceuticals.

ACKNOWLEDGEMENTS:

The authors are grateful to Head, Department of Chemistry and Principal, Nizam College for providing facilities.

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Received on 25.05.2011 Modified on 05.06.2011

Asian J. Research Chem. 4(8): August, 2011; Page 1297-1301