INTRODUCTION:

Ciprofloxacin is a fluroquinolone derivative. It is 1- cyclopropyl 1-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline¹. The Fluoroquinolones inhibit the enzyme bacterial DNA gyrase, which nicks double standard DNA, introduces negative supercoils and then reseals the nicked ends. This is necessary to prevent excessive positive supercoiling of the strands when they separate to permit replication or transcription. Structure of ciprofloxacin in shown in figure 1. The drug Ciprofloxacin is official in I.P², B.P³, and U.S.P⁴. Review of literature^5-10 reveals that no method is described for determination of ciprofloxacin by UV difference spectroscopy. The present papers describes difference spectroscopy method for the determination of ciprofloxacin as API and pharmaceutical dosage form

EXPERIMENTAL METHOD:

Preparation of standard stock solution of Ciprofloxacin

Accurately about 100 mg of the drug was weighed and transferred to a 100 ml volumetric flask and dissolved in about 15 ml of distilled water. The volume was then made up to the mark with distilled water. 2 ml of this drug solution was transferred to a 50 ml volumetric flask and further diluted up to 50 ml mark with 0.1N NaOH solution. Similarly, 2 ml of the drug solution was transferred to another 50 ml volumetric flask and further diluted up to 50 ml mark with 0.1N HCL solution. These solutions contained 40μg/ml of drug .

Determination of wavelength of maximum absorbance in 0.1N NaOH and 0.1N HCL.

2.5ml of the standard stock solution was pipette out and transferred to a 10 ml volumetric flask .The volume was then made up to the mark with 0.1N NaOH solution. This solution contained 10µg/ml of the drug. The absorbance of this solution was scanned in the U.V Range of 200 to 400 nm against 0.1N NaOH as blank. The maximum absorbance of Ciprofloxacin was obtained in the UV range of 272nm as shown in figure 2.

Figure 1: Chemical structure of ciprofloxacin

2.5ml of the standard stock solution was pipette out and transferred to a 10ml volumetric flask. The volume was then made up to the mark with 0.1N HCL solution. This solution contained 10µg/ml of the drug. The absorbance of this solution was scanned in the UV range of 200 to 400nm against 0.1N HCL as blank. The maximum absorbance of Ciprofloxacin was obtained in the UV range of 278 nm as shown in figure 3.

Preparation of calibration curve for Ciprofloxacin at 278 nm

0.5, 1.0, 1.5, 2, 2.5, 3, 3.5, 4.0, 4.5, 5.0 and 5.5, ml of standard stock solution of Ciprofloxacin, separately were pipette out in to a series of 10 ml volumetric flask. The volumes were made up to the mark with 0.1N NaOH and 0.1N HCL, separately and mixed to obtain solution in the conc. range of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 μg/ml of the drug.

Figure 2: Scan of Ciprofloxacin in the range of 200to 400nm in 0.1N NaOH.

Figure 3: Scan of Ciprofloxacin in the range of 200to 400nm in 0.1N HCL

The absorbances of these resultant solutions were measured at 278nm by placing 0.1N HCL solution in the reference cell and 0.1N NaOH solution in the sample cell and a graph was plotted between absorbance obtained and the concentrations of the solutions. The calibration curve was shown in figure 4. The linearity, slope, intercept, correlation coefficient and optical characteristics are summarized in Table1.

Recovery Studies and Validation of the Method according to ICH Guidelines¹¹

To study the accuracy of the above proposed method, recovery studies were carried out by the addition of the standard drug solution to the placebo and recovery of drug was calculated. Result of recovery studies are summarized in Table 3. Precision of the method was studied by carrying out interday and intraday analysis and is expressed as relative standard deviation. Specificity was checked by spiking reference standard by placebo. The results were found to be satisfactory and are reported in Table 3.

Table 1: Data for Calibration Curve of Ciprofloxacin

Concentration(µg/ml)	Absorbance^*
2	0.043
4	0.137
6	0.228
8	0.31
10	0.393
12	0.459
14	0.546
16	0.629
18	0.724
20	0.807
22	0.89

Table 2: Optical parameters and Regression Characteristics of Ciprofloxacin

Parameters	Observations
Beers law limit (µg/ml)	2-22
Molar absorptivity (1 mole^-1cm^-1)	1.44×10⁴
Sandell ssensitivity (mg/cm²/.001absorbance unit)	2.69×10^-2
Regression equation (y = a+bc)
Slope(b)	0.0417
Intercept(a)	0.0319
Correlation coefficient(r²)	0.999

Table 3: Summary of validation parameters

Validation Parameters	Observations
Linearity and Range (μg/ml)	2-22 (μg/ml)
Correlation Coefficient (r)	0.9993
Precision (RSD)
Repeatability (n=6)	1.39
Intraday (n=3)	1.08
Interday (n=3)	1.73
% Recovery	100.1
Specificity	No interference found

Estimation of Ciprofloxacin in tablet dosage forms:

The powder of 10 tablets of same batch number were mixed and accurately weighed. Accurately about 15 mg powder (equivalent to 10 mg of Ciprofloxacin) was dissolved in about 60 ml 0.1N HCL and 0.1 N NaOH, respectively dissolved and filtered through Whatmann Filter paper 40 into 100 ml volumetric flasks. The residue was washed with more solution into the flask. The volume was then made up to the mark with 0.1N HCL and 0.1N NaOH, respectively. 1ml of the resultant solution was transferred to a 10 ml volumetric flask and volume was made up to the mark with

0.1N HCL and 0.1 N NaOH solutions, respectively to obtain a solution of 10 μg/ml of the drug. The absorbance of these solutions was measured at 278 nm using 0.1N HCL as a blank. The concentrations of Ciprofloxacin present in tablet dosage forms were determined and are tabulated in Table 4.

Figure 4: Calibration curve of Ciprofloxacin at 278 nm.

SUPPLEMENTRY INFORMATION:

Instruments:

1) UV-Visible spectrophotometer, UV-210 (Elico)

2) Weighing balance, HR 200 (Shimadzu)

3) Ultra sonic bath, SW 45 (Toshcon/ Tosniwal)

Reagents:

Sodium Hydroxide, Hydrochloric Acid AR (Merck Limited), Water, distilled (In house produced)

RESULT AND DISCUSSION:

A simple and sensitive spectrophotometric method for the quantitative determination of Ciprofloxacin in either pure form or in pharmaceutical formulation was developed. Ciprofloxacin showed maximum absorption at 272nm in 0.1N HCL and 278nm in 0.1N NaOH solution, respectively. Spectrophotometric method linear response obtained was in the concentration range of 2-22 mg/ml with correlation coefficient 0.9993, recovery of the drug was found to be 100.1% and relative standard deviation was found to be less than 2 % for precision studies. The method was statistically validated according to ICH guidelines. The developed validated methods are simple, rapid, precise and accurate. The newly developed methods can be used for routine analysis of Ciprofloxacin in tablet dosage forms.

ACKNOWLEDGEMENTS:

Authors are sincerely thankful to Department of Pharmaceutical Science S.G.R.R.I.T.S Dehradun for providing necessary facilities and Zydus Cadila, Changodar, Ahmedabad Plant, for providing pure ciprofloxacin drug sample.

RFERENCCES:

1. Tripathi K P Essential of Medicinal Pharmacology, 4^th Ed.,1999, Jaypee Brothers Medical Publishers(P) Ltd:539-555.

2. Indian Pharmacopoeia Ministry of Health New Delhi, 1996,Vol.II, 76.

3. British Pharmacopoeia, I^st Ed., Vol.II, Her Majesty Office London, U.K, 88.

4. United State of Pharmacopoeia, Asian 3^rd Ed., 1997, US Pharmacopoeial convention, Inc., 28.

5. Groeneveld Pascal R Perez P (1985). Developed saimple sensitive method for analysis of Ciprofloxacin, Norfloxacin, Ofloxacin and Perfloxacin in serum.; J. Microchemical. 80: 1620-1635.

6. Marika, K Kimiko T Tsutomu K Koichi N (1998). Development of a simple, sensitive isocratic method for the detection and quantification of Ciprofloxacin in plasma and urine using reversed-phase HPLC with U.V detection. Oita . Japan. 44: 1251-1255.

7. Misabel, P Reguera, G Perez P (2004) Development of a simple and inexpensive method for the determination of ciprofloxacin using solid-phase spectrophotometry. J. Microchemical. 77: 79-84.

8. Najla, M Anil. K. S Erika R Maria K. Maria. (2005). Development and Validation of an Analytical method for quantitative determination of Ciprofloxacin and Norfloxacin in pharmaceutical preparations. Revista Brasileira de Ciencias Farmaceuticals. .41; 4; 122-127.

9. Jan. K., Urszula H., Justyna S., (2005). Development a Thin Layer Chromatography densitometric method development for identification and quantification of ciprofloxacin and an ethylenediamine compound, a desfluoro compound, by compound A and fluoroquinolonic acid as ciprofloxacin degradation products in pharmaceutical preparations. J. of AOAC International. 88; 1530-1536.

10. Aksoy B., Tsutomu, K., Antonia M., (2007). Developed and validated simple sensitive precise and stability indicatying reversed phase HPLC method for the dfetermination of Ciprofloxacin HCl in pharmaceutical dosage forms in the presence of its potential impurities; J. of AOAC International, 75;1432-1440.

11. Text on Validation of Analytical Procedures Q2B in; I.C.H. Harmonized Tripartite Guidelines; NOV. 1996.

Received on 14.05.2009 Modified on 18.07.2009

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 780-782