INTRODUCTION:

Ciprofloxacin is, chemically, 1-cyclopropyl-6-fluoro-1, 4- dihydro-4-oxo- 7 (piperazin-1-yl) quinoline-3-carboxylic acid¹; and belongs to the group of synthetic fluoroquinolone antibiotics with broad antimicrobial activity. It is believed that the mode of action of this family of drugs is through binding DNA-gyrase enzyme.

Fig 1: Structure of Ciprofloxacin

Ciprofloxacin is used in the treatment of urinary tract infections, gonorrhoea, chancroid, typhoid, meningitis, conjunctivitis, osteomyetis and joint infections, bacterial gastroenteritis. It has also been used to reduce stool volume in cholera. It is also used in prophylaxis of infections in cancer patients^2-3. The drug is official in British Pharmacopoeia, Indian Pharmacopoeia and United States Pharmacopoeia which describe a high performance liquid chromatographic (HPLC) method for its assay.

Assay of Ciprofloxacin in pharmaceuticals has previously been achieved by several analytical techniques^4-26such as HPLC, Spectrophotometry, fluorimetry, spectrofluorimetry, chemiluminometry, ISE-based potentiometry and voltammetry. However, many of these require expensive equipment and skilledoperation. The aim of present study work is to develop the method based on the ion pair extraction spectrophotometry (colorimetry) using methyl orange.

MATERIALS AND METHODS:

Instrument used: A Shimadzu UV-Vis Double beam spectrophotometer (Pharmaspec-1700) with 1cm matched quartz cells was used for all spectral measurements.

Table 1: Linearity data of ciprofloxacin
S. No.	Concentration (µg/mL)	Absorbance
1	0	0
2	100	0.211
3	200	0.414
4	300	0.616
5	400	0.813
6	500	0.999
7	600	1.205
8	700	1.43

Materials: The tablets (referred as CIPROLET®) were purchased from a local pharmacy (The label claim contained 500 mg of Ciprofloxacin).

All chemicals used were of analytical reagent grade and distilled water was used to prepare all solutions. Various chemicals used were Chloroform, Methyl orange, anhydrous sodium acetate, and Glacial acetic acid.

Preparation of acetate buffer (pH 2.8): 4 g of anhydrous sodium acetate was transferred intoa 1000 mL volumetric flask. 740 mL of distilled water and 155 mL of glacial acetic acid were added into the flask. Then the solution was diluted up to 1000 ml with distilled water. The pH of the solution was measured by pH meter. The pH of the buffer was found to be 2.8.

Preparation of methyl orange (0.5%): 0.5 g of methyl orange was weighed and dissolved in distilled water, sonicated for 2 minutes and final volume was made up to 100 mL with distilled water.

Fig 2: Calibration curve of Ciprofloxacin

Preparation of standard stock solution: 100 mg of ciprofloxacin hydrochloride was accurately weighed and transferred into a 100 mL volumetric flask containing 40mL of acetate buffer (pH 2.8) and sonnicated for 4 minutes. Final volume was made up to 100 mL with buffer in order to get 1mg/mL.

Preparation of calibration curve: 1mL, 2mL, 3mL, 4mL, 5mL, 6mL and 7mL of standard stock solutions were taken in 7 different 10 mL volumetric flasks and diluted up to mark with acetate buffer (pH 2.8). Then content of the volumetric flasks were transferred into seven different 125mL separating funnels. Four mL of methyl orange solution (0.5%) were added to each separating funnel. Then 15 mL of chloroform were added in each separating funnel and shaken well for 3 minutes and then kept for another 5mins. Then the drug was extracted into the chloroform layer and separated into seven different 50 mL volumetric flasks. Then the blank solution was prepared by utilising all the above reagents excluding the drug solution. Then the maximum absorbance was measured and it was found 429 nm. Then the calibration curve was constructed using concentration vs. absorbance. The linearity was found between 100 to 700 µg/mL of Ciprofloxacin hydrochloride. The Linearity data is given below in Table-1. The calibration curve of drug is shown in the below Fig- 2.

Analysis of Pharmaceutical formulations: Twenty tablets of ciprofloxacin hydrochloride were weighed and ground to fine powder. The solution was then filtered through Whatmann paper. Sixty seven mg of tablet powder equivalent to 50 mg of pure drug was weighed and dissolved in 10 mL of acetate buffer in a 50 mL volumetric flask and sonnicated and diluted up to mark using acetate buffer. Then 2 mL of the above solution was transferred into a 10mL volumetric flask and diluted up to 10 mL in order to get 200µg/mL and then it was transferred into a 125mL separating funnel. 4 mL of methyl orange solution (0.5%) and 15 mL chloroform were added into the separating funnel and shake for 3mins and kept for 5minutes. Then the drug was extracted and absorbance was measured against a reagent blank at 429 nm. The amount of Ciprofloxacin was determined by using the linear regression equation. The results of analysis of tablet are given in the Table- 2

SD: Standard deviation, SE: standard error, C.I.: Confidence Interval within which true value may be found at 95% confidence level = R ± ts/√n, R: Mean percent result of analysis of Recovery study (n = 5); Theoretical ‘t’ value at 95% confidence level for n - 1 degrees of freedom t (0.05, 4) = 2.77.

Method validation: As per ICH guide lines.

Linearity: The linearity range was found in between 100-700 µg/ml of Ciprofloxacin. The linear regression equation was found to be Y = 0.002 x + 0.0055, and Correlation coefficient r² = 0.9997.

Precision: Precision study was performed to find out intraday (within a day) variation in the estimation of ciprofloxacin at three different concentrations with the proposed method. Standard deviation (S.D.) was found to be less than 0.5 and the colour complex was stable and produces precise absorbance values. The colour complex was stable for a period of 8 hrs. The precision data is given in Table 3:

Table No. 3 Precision data of ciprofloxacin

S. No.	Conc.	Absorbance*	SD
1	200	0.4123	0.0061
2	400	0.818	0.0101
3	600	1.1987	0.0107

*Average of four determination

Accuracy: It was found out by recovery study using standard addition method. Known amounts of standard Ciprofloxacin was added to pre-analysed tablet samples at a level from 80% up to 120% and then subjected to the proposed colorimetric method. Results of recovery studies are shown in Table 4.

SD: Standard deviation, SE: standard error, C.I.: Confidence Interval within which true value may be found at 95% confidence level = R ± ts/√n, R: Mean percent result of analysis of Recovery study (n = 5). Theoretical‘t’ values at 95% confidence level for n - 1 degrees of freedom t (0.05, 4) = 2.77.

Sensitivity: Sandel’s sensitivity (µgcm^-3AU): 0.489140

Stability of complex: The drug and dye complex was stable for a period of 25 hours.

Mechanism of the Reaction: The studied fluoroquinolones contain terminal nitrogenatom in Piperazine moiety. In proper acidic medium, thisnitrogen atom is protonated to give positively charged quaternary ammonium group which in turn forms an ionpair complex with negatively charged dye containingsulphonic acid group. This complex is readily extractable inchloroform and measured at the appropriatewavelength. The theoretical basis of this method is that thedissociation equilibrium of BA-type (which is dissociated inaqueous medium) can be shifted toward the left (association) if the ion pair is removed by extraction using a solvent immiscible with water.

BA B⁺ + A^-

Where B⁺is the protonated amine drug and A^-is the dye anion form. The proposed reaction mechanism of the formation of ion-pair complex is shown in the Fig 3.

RESULT AND DISCUSSION:

In aqueous acidic medium, Ciprofloxacin forms a yellow absorbed ion-associated complex with methyl orange. A complex was extractable with chloroform. The analytical wavelength for measuring absorption maximum for Ciprofloxacin-methyl orange yellow complex was observed at 429 nm against the reagent blank. Absorption minimum at 429 nm observed for the reagent blank identical experimental conditions were used. The extent of formation of ion-associated Ciprofloxacin complex is governed by methyl orange concentration. The solute absorbances were plotted as a function of methyl orange concentration. The absorbance of the complexes initially increased in the concentration range of (0.02-0.5%) methyl orange and then attained practically a constant value in the concentration of (0.5%) methyl orange. Thus it was found that 4 ml of 0.5% concentration of methyl orange and 4ml of acetate buffer (pH 2.8) were necessary for the achievement of maximum colour intensity. Hence 4.0 ml of methyl orange and 2.0 ml of acetate buffer were selected. The optimum pH where the ion-associated complex shows maximum absorbance was found to be 2.8 in an experiment where it was mixed with the drug in aqueous solutions of varying pH 2-3. The decrease in absorbance beyond pH 2.8 is due to the new ion associated complex formation. The absorbance of reagent blank does not show any change with increasing pH. Hence a pH of 2.8 was used in all the subsequent experimental work. The effect of temperature on the product was studied at different temperatures. The coloured product was stable in the temperature range of 0.0-35 ⁰C. At higher temperatures the drug concentration is increased on prolonged heating due to volatile nature of chloroform. As a result, the absorbance value of the colored products was increased. However, resultant product was stable for a period of 8 hours at 25±5⁰C. The validity of the method for the assay of tablets was determined. The results of analysis of average recoveries obtained in each instance were compared with 100 percent theoretical value of Students‘t’ test. As the calculated‘t’ values are less than theoretical‘t’ values, it is concluded that the results of recoveries obtained are in accordance with 100 percent for each analyte. So the percentage recovery experiments revealed good accuracy of the data. There is no need for the separation of soluble excipients present in marketed tablets as the results were always reproducible equivalent to the labeled contents of the preparations. The molar ratio of the drug: dye was determined by Job’s method and found to be 1:2. The recovery results of the proposed method were well agreed with the official and reported RP-HPLC method for Ciprofloxacin tablets.

CONCLUSION:

A simple UV spectrophotometric method was developed for the determination of Ciprofloxacin in bulk and its pharmaceutical formulations. In this method ciprofloxacin was extracted by ion pair extraction with chloroform by treating with methyl orange and measuring absorbance at 429 nm. Therefore it was concluded that analytical methods developed for the selected drug was simple, selective, precise, accurate, economy and specific. The method had been validated statistically and by recovery study. The proposed method can be used as alternative method to the reported ones for the routine determination of selected drugs under the study (i.e. Ciprofloxacin) in bulk and pharmaceutical dosage forms.

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Received on 20.03.2012 Modified on 29.03.2012

Asian J. Research Chem. 5(4): April 2012; Page 537-540

Table No. 4 Recovery study data of Ciprofloxacin
% Level of recovery	Formulation (µg/ml)	Amount of Pure drug added (µg/ml)	Amount Of Drug found (µg/ml)	C.I.	SD	SE	t
80	200	160	360.384	100.1064 ±0.463	0.373	0.166	0.637
100	200	200	400.854	100.2135 ±0.541	0.435	0.194	1.095
120	200	240	440.826	100.1875 ±0.502	0.404	0.181	1.035