Stability Indicating RP-HPLC Method for Analysis of Ciprofloxacin in Tablet Dosage Form

Bhupendra Shrestha¹*, Hema Basnett¹, Sita Sharan Patel¹, Rashmi Karki¹, Shivani Agarwal¹, R. Mazumder² and B. Stephenrathinaraj³,

¹Department of Pharmaceutical Chemistry, Himalayan Pharmacy Institute, Majhitar, E.Sikkim-737136.

²Dept. of Pharmaceutics, Calcutta Institute of Pharmaceutical Technology and AHS, Howrah, West Bengal-711316. ³Dept. of Pharmaceutical Analysis, Vaagdevi College of Pharmacy, Hanamkonda, Warangal, Andhra Pradesh.

*Corresponding Author E-mail: shrestha2k@yahoo.com

ABSTRACT:

A simple, sensitive, accurate, reproducible and stability indicating RP-HPLC method was developed for the rapid analysis of Ciprofloxacin in tablet dosage form. The chromatographic separation was performed on a RP C18 (250 x 4.6 i.d) mm, 5µm, column. The mobile phase of the method consists of equal amount of acetonitrile and methanol along with 1% acetic acid in aqueous solution in the ratio of 8:8:84 v/v in the flow rate of 1.5 mL/min. The retention time for ciprofloxacin is found to be 7.7min when monitored at 280 nm. The detector response was linear in the concentration range of 200-700 µg/mL. The limit of detection and limit of quantification was 15.145 and 44.784 µg/mL respectively. The percentage assay of Ciprofloxacin was 99.92%. Correlation coefficient (r) of the regression equation is more than 0.999. The precision of the method was established by intra and inter day assay values which were less than 1%. None of the degraded products interfered with the Ciprofloxacin peak thus ensuring stability indicating nature of the method.

KEYWORDS: Ciprofloxacin, Stability Indicating, RP-HPLC

INTRODUCTION:

Antibiotics play a pivotal role to check pathogenic bacterial born diseases and are one of the extensively used drugs throughout the world but more so in the developing countries. Development of antibiotic resistance in bacteria continuously incites the scientists to modify the existing drugs or to develop newer remedies¹. Ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-(1-piperazinyl)-3-quinoline carboxylic acid) which is structurally related to nalidixic acid, is one of the very popular members of the flouroquinolones group of antibiotics. It is a synthetic antibiotic which acts by selectively inhibiting the enzyme DNA gyrase, which is necessary for the synthesis of bacterial DNA. These are most frequently used for the treatment of urinary, pulmonary, and digestive infections². The Minimum inhibitory concentration of ciprofloxacin against gram negative bacilli is <0.1ug/mL while gram positive bacteria are inhibited at relatively higher concentrations³.

A literature survey reveals some HPLC methods reported for the determination of ciprofloxacin in biological fluids^4-7. Mansilla et al. describes an analytical method for enoxacin, ciprofloxacin, norfloxacin and ofloxacin with photoinduced fluorimetric (PIF) detection⁸. Ballesteros et al. has reported LC–ESI–MS method for identification and simultaneous determination of ciprofloxacin, ofloxacin and norfloxacin in human urine after extraction of the fluoroquinolones by SPE⁹. Amini et al. presented a method for determination of ciprofloxacin by HPLC in human plasma¹⁰.

All the methods described above are not validated for its performance under stress condition so rendering them unsuitable for stability study samples. Therefore an attempt was made to develop a new, simple, sensitive and rapid stability indicating HPLC method for the determination of ciprofloxacin HCl in the presence of its degraded products as well. The method was validated according to procedures and acceptance criteria of FDA and ICH guidelines. The method can be suitably used for analysis of ciprofloxacin tablets as well.

EXPERIMENTAL

Ciprofloxacin was obtained as gift sample from Zydus Pharmaceuticals. Methanol and acetonitrile used were of HPLC grade and purchased from S.D Fine Chem Ltd, Mumbai. HPLC grade water was used and acetic acid used was of AR grade. Commercially available tablets Cifran (500mg, Ranbaxy) were procured from local market.

TABLE 1 : Analysis of Tablet Formulation

Component

Amount Present

(mg)

Amount Found* %

Standard Deviation

% RSD

Ciprofloxacin

500

99.92

0.5994

0.5998

*Average of six determinations

TABLE 2: Validation and System Suitability Studies

Parameter	Results
Retention time(min)	7.7
Tailing factor	1.10
Theoretical plates	8756
Linearity range(µg/ml)	200-700
Intercept	109.56
Slope	48.76
Correlation coefficient	1.0
Limit of detection(µg/ml)	15.145
Limit of quantitation(µg/ml)	44.784
Mean % Recovery	99.75

Instrument

Shimadzu LC-20 AT liquid chromatography in isocratic mode with Prominence SPD-20A UV-Visible detector was used for quantitative HPLC determination. The HPLC system was equipped with the Spinchrom (Shimadzu) software. Sartorius(CP225D) electronic balance was used for weighing the materials.

Chromatographic condition

Mobile phase used for the analysis consist of acetonitrile, methanol and 1% acetic acid in aqueous solution in the ratio of 8:8:84 v/v. They were filtered before use through a 0.45 µm membrane filter and pumped through the column RP C18 (250 x 4.6 i.d)mm, 5µm, in isocratic mode at a flow rate of 1.5 mL/min. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The analysis was performed at ambient temperature and the run time was set at 10 min. The eluents were monitored at 280 nm and retention of ciprofloxacin was found to be 7.7 min.

Preparation of Standard Solution

About 50 mg of standard ciprofloxacin hydrochloride was accurately weighed and transferred to a 100 ml volumetric flask, 25 ml of mobile phase was added, sonicated for 15 min and the volume was made up with the mobile phase to get a concentration of 500 µg/mL.

Preparation of Sample Solution

Twenty tablets (Cifran) were weighed accurately and triturated to fine powder. A quantity equivalent to 50 mg of ciprofloxacin was transferred into a 100 ml volumetric flask, 25 ml of mobile phase was added, sonicated for 15 min and the volume made up with the mobile phase. The solution was filtered through a 0.45 µm membrane filter before injecting into the system.

Ciprofloxacin Hydrochloride

Assay

Sample solution (20 μl) was injected into the liquid chromatograph. The retention time was found to be 7.7 minutes. The amount of drug present per tablet was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in Table 1.

Validation of the Method

The aim of method validation was to confirm that the present method was suitable for its intended purpose as described in ICH guidelines Q2A and Q2B^11,12. The described method has been extensively validated in terms of linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), specificity and system suitability. The precision was expressed with respect to the intra-and inter-day variation in the expected drug concentrations. The accuracy was expressed in terms of percent recovery. After validation, the developed method has been applied to analysis of ciprofloxacin HCl tablets.

System Suitability

The system suitability was determined by five replicate injections from freshly prepared standard solutions and analyzing for their peak area, theoretical plates (N) and tailing factors (T). The results of the system suitability test in comparison with the required limits are shown in Table 2.According to the results presented, the proposed method fulfills these requirements within the accepted limits.

Linearity

Standard Ciprofloxacin was diluted with the mobile phase such that the final concentrations of 200-700µg/mL was obtained. Each of these drug solutions (20 µl) was injected three times into the column, and the peak areas and retention times were recorded. The correlation coefficient obtained was 1.0.

Precision

The precision of the proposed method were assessed as repeatability and intermediate precision performing six replicate injections of the sample solutions at 100% level of the test concentration. The RSD value of the measurement was 0.214% confirming good precision of the proposed method.

Accuracy

Accuracy of the proposed method was established by recovery experiments. This study was employed by addition of known amounts of ciprofloxacin HCl onto the tablet matrix. The resulting mixtures were analyzed as described for tablets. Results obtained from recovery studies are given in Table 3.

Limit of Detection and Limit of Quantitation

In the present study, the LOD and LOQ were based on the third approach and were calculated according to the 3.3σ/s and 10 σ/s criterions, respectively; where σ is the standard deviation of the peak areas and s is the slope of the corresponding calibration curve. The LOD and LOQ values of the developed method are presented in Table 2.

Stress Studies

Acid-base, thermal and photo degradation studies were carried out for the powdered sample of ciprofloxacin. For acid and base degradation, powdered tablet samples were kept in 0.1 N sodium hydroxide or 0.1 N methanolic hydrochloric acid for 24 hours. For photo and thermal decomposition experiments, tablets containing 500 mg of ciprofloxacin hydrochloride were exposed to ultraviolet light at 254 nm and dry heat at 60 °C separately for 24 h. The results were compared with the assay value.

RESULTS AND DISCUSSION

The HPLC method developed here is one which can be used in the routine analysis of ciprofloxacin HCl in the tablet dosage form. The method was validated for different parameters and it passed all of them. The stability indicating nature of the method was established by degradation studies. It was found that the sample degraded in all the parameters tested to some extent except in acid degradation but none of the degraded products interfered with the analysis of ciprofloxacin.

REFERENCES :

1. Muhammad K.K., Muhammad F.K., Assessment of Bioequivalence of Ciprofloxacin in healthy male subjects using HPLC, Pak. J. Pharm. Sci., July 2008, 21(3), 299-306.

2. Samanidou V.F., Christodoulou E.A., Papadoyannis I.N., Cur. Pharm. Analysis, 2005, 1, 155 – 193.

3. K.D. Tripathi, Essentials of Medical Pharmacology, 5^th Edn., Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, 2004, 648.

4. Kamberi M, Tsutsumi K, Kotegawa T, Nakamura K, Nakano S, Determination of ciprofloxacin in plasma and urine by HPLC with ultraviolet detection, Clin. Chem., Feb 1998, 44:1251–1255

5. Maya M.T., Goncalves N.J., Silva N.B., Morais J.A., J. Chromatogr. B 2001, 755(1-2):305–309.

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7. Imre S, Dogaru M.T., Vari C.E., Muntean T, Kelemen L, J. Pharm. Biomed. Anal. 2003, 33:125–130.

8. Mansilla E.A., Pena M.A., Gomez G.D, Salinas F, HPLC determination of enoxacin, ciprofloxacin, norfloxacin and ofloxacin with photoinduced fluorimetric detection and multiemission scanning - Application to urine and serum, J. of Chromatogr. B, May 2005, 822 : 2005, 185–193.

9. Ballesteros O, Toro I, Sanz N. V., Navalon A, Vilchez J.L., Barbosa J, J. Chromatogr. B 2003, 798:137–144.

10. Amini M, Khanavi M, Shafiee A, Simple High-Performance Liquid Chromatographic Method for Determination of Ciprofloxacin in Human Plasma, Iran. J. of Ph. Res., June 2004, 2: 99-101.

11. ICH Q2A, Text on Validation of Analytical Methods, Definition and Terminology(International Conference on Harmonisation of Technical requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland),1995.

12. ICH Q2B Validation of Analytical Procedures, Methodology (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland), 1997.

Received on 11.09.2009 Modified on 08.11.2009

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 221-223

Level of % Recovery	Component	Amount present (mg)	Amount of standard added(mg)	% Recovery*	%RSD
80	Cipro	500	400	99.94	0.276
100	Cipro	500	500	99.28	0.850
120	Cipro	500	600	100.05	0.250