Estimation of Ciprofloxacin Hydrochloride in Bulk and Formulation by Derivative UV-Spectrophotometric Methods

Simple, fast and reliable spectrophotometric methods were developed for determination of Ciprofloxacin Hydrochloride in bulk and pharmaceutical dosage forms. The solutions of standard and the sample were prepared in Distilled Water. The quantitative determination of the drug was carried out using the zero/0 th , first, and second order method values measured at264, 273and 273 nm respectively. Calibration graphs constructed at their wavelengths of determination were linear in the concentration range of Ciprofloxacin Hydrochloride using 2-10μg/ml (r2=0.9991, r2=0.9993, r2=0.9955) for zero, first and second order spectrophotometric method. All the proposed methods have been extensively validated as per ICH guidelines. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations. The developed methods were successfully applied to estimate the amount of Ciprofloxacin Hydrochloride in pharmaceutical formulations.


Derivative Spectrophotometric Methods.
Derivative spectrophotometry is a useful means of resolving two overlapping spectra and eliminating matrix interferences or interferences due to an indistinct shoulder on side of an absorption band. Derivative spectrophotometry involves the conversion of a normal spectrum to its first, second or higher derivative spectrum. In the context of derivative spectrophotometry, the normal absorption spectrum is referred to as the fundamental, zero th order or D 0 spectrum. The absorbance of a sample is differentiated with respect to wavelength λ to generate first, second or higher order derivative.
[A]= f(λ): zero order [dA/dλ= f(λ ): first order [d 2 A/dλ 2 ]= f(λ): second order The first derivative spectrum of an absorption band is characterized by a maximum, a minimum, and a cross-over point at the λ max of the absorption band. The second derivative spectrum is characterized by two satellite maxima and an inverted band of which the minimum corresponds to the λ max of the fundamental band [26].

Apparatus and instrumentation
A shimadzu 1800 UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. Single Pan Electronic balance (CONTECH, CA 223, India) was used for weighing purpose. Sonication of the solutions was carried out using an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India). Calibrated volumetric glassware (Borosil®) was used for the validation study.

Materials
Reference standard of Ciprofloxacin Hydrochloride API was supplied as gift sample by Marksan Pharmaceutical Ltd., Verna, and Goa. Tablet sample with label claim 500 mg per tablet were purchased from local market Pune.

Method development 2.4.1 Preparation of Standard and Sample Solutions
Stock solution of 10μg/ml of Ciprofloxacin Hydrochloride was prepared in Distilled Water, for zero, first and second order spectrophotometric analysis. The standard solutions were prepared by dilution of the stock solution with Distilled water in a concentration range of 2, 4, 6, 8, and 10μg/ml with Distilled water for zero order and area under the curve spectrophotometric methods. Distilled water was used as a blank solution.

Assay Procedure
Twenty tablets each containing 500 mg of Ciprofloxacin Hydrochloride were weighed crushed to powder and average weight was calculated. Powder equivalent to 10 mg of Ciprofloxacin Hydrochloride was transferred in 100ml of volumetric flask. A 50 ml of distilled water was added and sonicated for 15 minutes. Then solution was further diluted up to the mark with distilled water. The solution was filtered using Whatmann filter paper no. 41; first 5 ml of filtrate was discarded. This solution was further diluted to obtain 10µg/mL solution with water subjected for UV analysis using distilled water as blank. Appropriate dilutions were made with Distilled water from stock solution for zero, first and second order spectrophotometric methods.

Results and Discussion
The zero, first and second order method values spectra for Ciprofloxacin Hydrochloride were recorded at the wavelength of 272nm, 274nm, 274nm respectively.

Linearity and Range
Under the experimental conditions described, the graph obtained for zero, first and second order method spectra showed linear relationship. Regression analysis was made for the slope, intercept and correlation coefficient values. The regression equations of calibration curves were y=0.0411x+0.015 (r 2 =0.9991) at 272 nm for zero order derivative spectrophotometry, y=0.002x-0.0022 (r 2 =0.9993) at 274 nm for first order derivative spectrophotometry and y=0.0009x+0.0014 (r 2 =0.9955) at 274 nm for second order derivative spectrophotometry. The range was found to be 5-25μg/ml for all zero, first and second order spectrophotometric methods. www.ssjournals.com

Accuracy
To study the accuracy of the proposed methods, and to check the interference from excipients used in the dosage forms, recovery experiments were carried out by the standard addition method. The accuracy for the analytical method was evaluated at 80%, 100% and 120% levels of 20µg/ml standard solution. For Zero, first and second order derivative were measured in wavelength range at 272, 274 and 274nm respectively and results were obtained in terms of percent recovery. Three determinations at each level were performed and % RSD was calculated for each level.

Precision
To determine the precision of the method, Ciprofloxacin Hydrochloride solutions at a concentration of 10μg/ml were analysed each three times for all zero, first and second order spectrophotometric methods. Solutions for the standard curves were prepared fresh every day.

Sensitivity
The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula LOD = 3.3σ/S The Limit of Quantification (LOQ) is the smallest concentration of the analyte, which gives response that can be accurately quantified. LOQ was calculated using the following formula LOQ = 10σ/S Where, σ is standard deviation of the response and S is the slope of the calibration curve. The LOD and LOQ were found to be 0.40μg/ml and 1.21μg/ml for zero order derivative, 0.315µg/ml & 0.956µg/ml for first order derivative and 0.814µg/ml &2.462µg/ml for second order derivative respectively.

Analysis of the Marketed Formulation:
There was no interference from the excipients commonly present in the tablets. The drug content was found to be 99.58%, 99.10% and 99.20% for zero, first and second order derivative spectrophotometric methods respectively. It may therefore be inferred that degradation of Ciprofloxacin Hydrochloride had not occurred in the marketed formulations that were analysed by this method. The low % R.S.D. value indicated the suitability of this method for routine analysis of Ciprofloxacin Hydrochloride in pharmaceutical dosage form.

Conclusion
No UV/ zero, first and second order spectrophotometric methods have been described for the determination of Ciprofloxacin Hydrochloride. Therefore simple, fast and reliable derivative spectrophotometric methods were developed for the routine determination of Ciprofloxacin Hydrochloride. The developed methods can be concluded as accurate, sensitive and precise and can be easily applied to the pharmaceutical formulation.