Development and Validation of a StabilityIndicating HPLC Method for Determination of Ciprooxacin Hydrochloride and its Related Compounds in Film-Coated

Tablets
2007, 66, S57S63

B. Aksoy1, I. Ku u c kgu zel2,&, S. Rollas2

1 2

Biofarma Pharmaceutical Industry Co. Inc., Fatih Cad. No: 2, Kartal, 34885 Istanbul, Turkey Department of Pharmaceutical Chemistry, Marmara University Faculty of Pharmacy, Haydarpasa, 34668 Istanbul, Turkey; E-Mail: ikucukguzel@marmara.edu.tr

Received: 5 February 2007 / Revised: 11 April 2007 / Accepted: 7 May 2007 Online publication: 27 June 2007

Abstract
The objective of the current study was the development and subsequent validation of a simple, sensitive, precise and stability-indicating reversed-phase HPLC method for the determination of ciprooxacin HCl in pharmaceutical dosage forms in the presence of its potential impurities. The chromatographic separation of ciprooxacin HCl and its related compounds was achieved on an Inertsil ODS3 column using UV detection. The optimized mobile phase consisted of phosphoric acid solution: acetonitril. The proposed method provided linear responses within the concentration range 250750 lg mL)1 for ciprooxacin HCl and 0.51.5 lg mL)1 for its related compounds. LOD and LOQ values for the active substance were 5.159 and 15.632 lg mL)1, respectively. Correlation coefcients (r) of the regression equations for the impurities were greater than 0.99 in all cases. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed.

Keywords
Column liquid chromatography Stability-indicating method Film-coated tablets Ciprooxacin

Introduction
Ciprooxacin (1-cyclopropyl-6-uoro-1,4dihydro-4-oxo-(1-piperazinyl)-3-quinoline carboxylic acid) is a synthetic and broad spectrum uoroquinolone antibacterial

Application of Separation Techniques in Turkey

agent structurally related to nalidixic acid (Fig. 1). Ciprooxacin, however, demonstrated antimicrobial activity superior to that of nalidixic acid and noroxacin against all pathogens tested [1, 2]. Its mechanism of action is related to inhibition of bacterial DNA gyrase [3, 4]. Ciprooxacin has a broad antibacterial activity spectrum against most GramChromatographia Supplement Vol. 66, 2007

negative bacteria which are highly susceptible in vitro whereas many Gramnegative bacteria are susceptible or moderately susceptible [4, 5]. It is used in cases of urinary and respiratory tract infections as well as gonorrhea, osteomyelitis and infections of the bone, joints and gastro-intestinal system [4]. A literature survey reveals some HPLC methods reported for the determination of ciprooxacin in biological uids [612]. Liang et al. [13] reported separation and determination of ciprooxacin, cinoxacin, levooxacin, gatioxacin, moxioxacin and trovaoxacin by reversed-phase HPLC in human plasma using UV and uorescence detection; whereas, another report by Ballesteros et al. [14] describes an LCESIMS method for identication and simultaneous determination of ciprooxacin, ooxacin and noroxacin in human urine after extraction of the uoroquinolones by SPE. Separation of ve quinolones including ciprooxacin was also described using reversed phase HPLC with uorescence detection for the determination in food [15]. Other reports related to pharmaceuticals, include simultaneous RP-HPLC determination of ciprooxacin and metronidazole in intravenous admixtures [16]; RP-HPLC determination of ciprooxacin as bulk drug and in pharmaceutical dosage forms [17]; an HPTLC procedure for the determination and purity control of ciprooxacin HCl in coated tablets [18]; separation and determination of quinolones including

Original DOI: 10.1365/s10337-007-0287-6

S57

2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

O F COOH

HN N

O COOH

N HN

Cl

Ciprofloxacin HCl
1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-1,4dihydroquinoline-3-carboxylic acid.
O COOH

Impurity D
7-chloro-1-cyclopropyl-4-oxo-6-piperazin-1-yl-1,4dihydroquinoline-3-carboxylic acid.
O F

N HN

N
HN

Impurity B (Desfluoro compound)

1-cyclopropyl-4-oxo-7-piperazin-1-yl-1,4dihydroquinoline-3-carboxylic acid.
O F COOH

Impurity E (Descarboxy analogue)

1-cyclopropyl-6-fluoro-7-piperazin-1-ylquinolin-4(1H )one.
O Cl COOH

HN H2N

N HN

Impurity C (Ethylenediamine analogue)

7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro4-oxo-1,4-dihydroquinoline-3-carboxylic acid.

Chloro analogue
6-chloro-1-cyclopropyl-4-oxo-7-piperazin-1-yl-1,4dihydroquinoline-3-carboxylic acid.

Fig. 1. Chemical structures of ciprooxacin HCl, imp-B, imp-C, imp-D, imp-E and chloro analogue

Germany) 1100 series instrument equipped with a quaternary solvent delivery system, an Agilent series 1100 UV detector and Agilent 1100 G1322A model online degasser. An Agilent G1329A ALS autosampler with a 100 lL sample loop was used for the injection of analytes. Chromatographic data were collected and processed using Agilent Chemstation software. The separation was performed at ambient temperature, on a reversed phase Inertsil ODS3 C8 column (250 4.6 mm; 5 lm particle size). A pre-column packed with the same sorbent was used. All experiments were employed in the isocratic mode. The mobile phase was prepared by mixing 2.45 g of phosphoric acid with water followed by diluting it to 1 L. Before mixing with organic solvents, the nal pH of this solution was adjusted to pH 3.0 with TEA and 870 mL of this solution was mixed with 130 mL of acetonitrile. The mobile phase was ltered through Millipore 0.45 lm membrane lters and degassed by sonication. A ow rate of 1.5 mL min)1 was maintained. Injection volume was set to 10 lL for the assay method and 50 lL for the impurities method. UV detection of the analytes was carried out at 278 nm.

ciprooxacin in tablet formulations [19]. Lacroix et al. [20] reported an HPLC method for determination of ciprooxacin HCl and its related compounds in raw materials. Of these methods, only the one by Thoppil et al. [16] is stability indicating whereas, it cannot allow analysis of the impurities at the same time. An attempt was therefore made to develop a new, simple, sensitive, rapid and extensively validated HPLC method for the determination of ciprooxacin HCl in the presence of its related compounds. It was also aimed at developing the technique as a stability-indicating method providing assay of the active pharmaceutical ingredient (API) together with tracking of the impurities already present or as a result of forced degradation studies in lm-coated tablets.

analogue) (Fig. 1) were received from Dr. Reddys Laboratories Ltd, Hyderabad, India. The inactive ingredients used as the drug matrix include, colloidal silica, corn starch, croscarmelose sodium, crospovidone, hydroxypropyl methylcellulose, macrogol, magnesium stearate, microcrystalline cellulose and titaniumdioxide. Hydrochloric acid and sodium hydroxide were of analytical grade and supplied from Merck (Darmstadt, Germany). HPLC grade phosphoric acid, triethylamine, acetonitril and methanol were supplied from J.T.Baker (Deventer, The Netherlands). High purity water was prepared using Millipore Milli Q purication system (Molsheim, France). Cipro (500 mg ciprooxacin HCl) lmcoated tablets were kindly provided by Biofarma Pharmaceutical Industry Co. Inc. (Istanbul, Turkey).

Standard Stock Solutions and Construction of Calibration Curves

For the preparation of the stock solutions, 125 mg of ciprooxacin hydrochloride reference standard and 15.18 mg of placebo powder was weighed into a 100 mL volumetric ask and it was made up to volume with the mobile phase. Placebo powder was a mixture of colloidal silica, cornstarch, croscarmelose sodium, crospovidone, hydroxypropyl methylcellulose, macrogol, magnesium stearate, microcrystalline cellulose and titaniumdioxide, which were present in tablet matrix. These samples were designed to mimic the tablet formulation, in order to show that there was no interaction between the tablet matrix and analytes. For the calibration of the assay method, 2.0, 3.0, 4.0, 5.0 or 6.0 mL aliquots of the stock solution were diluted to 10 mL with the mobile phase to give the nal concentrations of 250, 375, 500, 625 and 750 lg mL)1. Stock solutions of the impurities (impB, imp-C, imp-D, imp-E and chloro Original

Experimental
Chemicals and Reagents
Ciprooxacin hydrochloride (as monohydrate) and its related compounds (impB, imp-C, imp-D, imp-E and chloro

Apparatus and Chromatographic Conditions

The liquid chromatographic system, used in the present study, consisted of an Agilent Technologies (Waldbronn, Chromatographia Supplement Vol. 66, 2007

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analogue) were prepared by dissolving 2.5 mg of the related compound in the mobile phase followed by dilution with the same solvent to 50 mL in a volumetric ask. For calibration of the impurities method, 1.0, 1.5, 2.0, 2.5 or 3.0 mL of each stock solution of the related compounds were transferred into a 100 mL volumetric ask, 50 mg of ciprooxacin HCl and 7.59 mg of placebo powder were added and this solution was made up to volume with the mobile phase. Calibration solutions for the impurities method contained each of the related compounds at concentrations ranging 0.5 1.5 lg mL)1, as well as the active and tablet matrix. The calibration curves for ciprooxacin HCl and its related compounds were constructed by plotting the peak areas against the analyte concentration.

rately for assay and impurities methods adjusting nal concentrations of the samples to 500 lg mL)1 for ciprooxacin HCl and 1.0 lg mL)1 for each of the related compounds. HPLC samples were then prepared and the resulting mixtures were analysed as described for pharmaceutical dosage forms.

Sample Preparations
Ten tablets were accurately weighed, their mean weight determined, and then nely powdered. An amount equivalent to 25 mg ciprooxacin HCl was transferred into a 50 mL volumetric ask, 40 mL of the mobile phase added, sonicated for 15 min and then made up to volume with the mobile phase. The amounts of ciprooxacin HCl and its related compounds in nished pharmaceutical dosage forms were individually calculated using the related linear regression equations.

Precision of the Method

Assay method precision was evaluated by carrying out six independent assays of test sample of ciprooxacin HCl at 100% level of the test concentration, 500 lg mL)1. The percentage of R.S.D. of six assay values obtained was calculated. The precision of the related substance method was checked by injecting six individual preparations of (500 lg mL)1) ciprooxacin HCl spiked with 0.2% each of imp-B, imp-C, imp-D, imp-E and chloro analogue (1.0 lg mL)1) with respect to analyte concentration. The percentage of R.S.D. of area for each of the impurities was calculated. The inter-day variation was similarly evaluated over a period of one week. The intermediate precision of the method was also evaluated by a different analyst and a different instrument in the same laboratory.

Accelerated Degradation Studies

All degradation experiments on lmcoated tablets were performed at a drug concentration of 500 lg mL)1. For acid and basic degradation, powdered tablet samples equivalent to 25 mg ciprooxacin HCl were weighed into a 50-mL volumetric ask, dissolved and made up to volume with 0.1 N sodium hydroxide or 0.1 N methanolic hydrochloric acid and the resulting solutions were kept for 72 h. For photo and thermal decomposition experiments, lm-coated tablets containing 500 mg of ciprooxacin hydrochloride were exposed to direct daylight, ultraviolet light at 254 nm, or dry heat at 60 C, for a study period of 24 h. Tablets were also analyzed at the time of beginning for control experiments.

Recovery Studies
To demonstrate the accuracy of the proposed method and to see whether there is interference from excipients used in the dosage forms, recovery studies were employed. This was carried out by adding known amounts of ciprooxacin HCl and its related compounds (imp-B, imp-C, imp-D, imp-E and chloro analogue) to the placebo powder consisting of drugmatrix components mentioned above. Recovery samples were prepared sepaOriginal

Results and Discussion

Optimization of the Separation Method
The HPLC conditions were optimized in order to provide an adequate separation of ciprooxacin HCl and its related compounds; imp-B, imp-C, imp-D, impE and the chloro analogue. Mobile phase and ow rate was selected according to peak parameters (peak height, tailing, Chromatographia Supplement Vol. 66, 2007

plate number), baseline smoothness, run time, ease of mobile phase preparation. The liquid chromatographic system with the mobile phase containing phosphoric acid solution (2.45 g L)1, adjusted to pH 3.0 with TEA) acetonitril (87:13, v/v) and 1.5 mL min)1 ow rate proved quite robust. According to European Pharmacopeia, suppl. 4.6 [21], an ODS column was recommended as the stationary phase for ciprooxacin HCl and its impurities. The use of C18 columns for quantitative analysis of ciprooxacin or other uoroquinolones in pharmaceutical and biological samples has been well documented [12, 13, 15]. It has also been reported that ODS columns show optimal performance within a pH 27 range for quinolone derivatives [22]. Therefore attempts were started with reversed-phase ODS columns, at rst with Luna C18 (250 4.6 mm, 5 mm) and Nucleosil C18 (250 4.6 mm, 5 mm). Due to the amphoteric nature of ciprooxacin and residual silanol groups present in the columns, tailing were observed as previously reported [13, 15]. When Prodigy ODS2 and Prodigy ODS3 columns (250 4.6 mm, 5 lm) were subsequently tried, better peak shapes were observed although tailing was still a problem, probably due to the presence of metal impurities. An Inertsil ODS3 column (250 4.6 mm, 5 lm) which is of high purity and base deactivated, has been recommended because of its demonstrated ruggedness and reproducibility in this assay. Optimum wavelength for detection of ciprooxacin and its related compounds was 278 nm at which satisfactory detector response for all analytes was obtained. Injection volume was set to 10 lL for assay whereas it was increased to 50 lL for the impurities method which requires higher sensitivity. Figure 2 shows a typical chromatogram obtained from the analysis of a standard solution of ciprooxacin HCl; whereas Fig. 3 depicts chromatographic prole obtained from the active substance spiked with imp-B, imp-C, imp-D, imp-E and chloro analogue, using the proposed method. As shown in Fig. 3, ciprooxacin and its related compounds were eluted, forming well-shaped, sharp and symmetrical peaks, well separated from the solvent front and each other. The retention times were 2.797, 4.872, 5.517, 7.546, 9.690 and 15.591 min for imp-E, imp-B, imp-C, ciprooxacin HCl, imp-D and chloro analogue, respectively.

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Fig. 2. A typical chromatogram obtained from the injection of a standard solution of ciprooxacin HCl (500 lg mL)1). Chromatographic conditions: column, Inertsil ODS3 C8 column (250 4.6 mm i.d., 5 lm particle size); isocratic elution with the mobile phase consisting of phosphoric acid solution (2.45 g L)1, adjusted to pH 3.0 with TEA)acetonitril (87:13, v/v); ow rate, 1.5 mL min)1; temperature, ambient; injection volume, 10 lL; UV detection, 278 nm

and they were used to verify that the proposed method was able to produce good resolution between the peaks of interest with high reproducibility [25]. The system suitability was determined by ve replicate injections from freshly prepared standard solutions and analyzing each solute for their peak area, theoretical plates (N), resolution (R) and tailing factors (T). System suitability requirements for the assay and impurity methods were an RSD less than 1%, peak resolution (R) greater than 2.0 between two adjacent peaks for three analytes, theoretical plate numbers (N) at least 2,000 for each peak and USP tailing factors (T) less than 1.5. The results of the system suitability test in comparison with the required limits are shown in Table 1. According to the results presented, the proposed method fullls these requirements within the accepted limits.

Linearity
Linear calibration plots for the ciprooxacin HCl assay method was obtained over the calibration range of 50150% of nominal concentration, 250750 lg mL)1 and the correlation coefcient obtained was 1.000 (n = 3). The results exhibit that an excellent correlation existed between the peak area and concentration of the analyte. Linear calibration plots for the impurity method were obtained over the calibration range of 50 150% of nominal concentration, 0.5 1.5 lg mL)1 for impurities B, C, D, E and chloro analogue. The correlation coefcient obtained was greater than 0.99 for each of the related substances (n = 3). The plot of peak areas versus concentrations of all analytes were found to be linear within the concentration ranges stated above. Results obtained from regression analysis of the linearity data for ciprooxacin and its related compounds are summarized in Table 2.

Fig. 3. HPLC prole obtained from ciprooxacin HCl (500 lg mL)1) spiked with its related compounds at 0.2% level (1 lg mL)1). Chromatographic conditions: as for Fig. 2 except injection volume was 50 lL. Peaks: 1 = imp-E, tR = 2.797 min; 2 = imp-B, tR = 4.872 min; 3 = imp-C, tR = 5.517 min; 4 = ciprooxacin HCl, tR = 7.546 min; 5 = imp-D, tR = 9.690 min; 6 = chloro analogue, tR = 15.591 min

Validation of the Method

The aim of method validation was to conrm that the present method was suitable for its intended purpose as described in ICH guidelines Q2A and Q2B [23, 24]. The described method has been extensively validated in terms of specicity, linearity, accuracy, precision, limits of detection (LOD) and quantication (LOQ), robustness and system suitability. The precision (% relative standard deviation) 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 of the known amount of drugs added to the known amount of the pharmaceutical dosage forms. After validation, the developed method has been applied to coated tablets containing 500 mg of ciprooxacin HCl.

Limit of Detection and Limit of Quantitation

The ICH guideline [24] describes several approaches to determine the detection and quantitation limits. These include visual evaluation, signal-to-noise ratio and the use of standard deviation of the response and the slope of the calibration curve. In the present study, the LOD and Original

System Suitability
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LOQ were based on the third approach and were calculated according to the 3.3r/s and 10r/s criterions, respectively; where r 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. The limit of detection for imp-B, imp-C, imp-D, impE and chloro analogue were calculated as 0.014, 0.023, 0.013, 0.014 and 0.020% (of analyte concentration, i.e. 500 lg mL)1), respectively. The LOQ for imp-B, imp-C, imp-D, imp-E and chloro analogue were calculated as 0.044, 0.071, 0.040, 0.043 and 0.060% (of analyte concentration, i.e. 500 lg mL)1), respectively (Table 2).

Table 1. System suitability results of the proposed method

Substance Impurity E Impurity B Impurity C Ciprooxacin HCl Impurity D Chloro analogue Required limits k 0.485 1.587 1.929 3.006 4.145 7.278 k > 1.5 T 1.248 1.101 1.089 1.306 0.932 1.041 T < 1.5 N 6,071 9,132 9,804 10,574 8,785 12,312 N > 2,000 R 6.915 11.942 3.025 4.386 3.844 12.097 R>2

Table 2. Results of regression analysis of the linearity data of ciprooxacin and its related compounds Substance Range (lg mL)1) 250750 0.51.5 0.51.5 0.51.5 0.51.5 0.51.5 Slope Intercept r2 LOD (lg mL)1) 5.159 0.072 0.117 0.066 0.071 0.099 LOQ (lg mL)1) 15.632 0.219 0.355 0.199 0.214 0.299

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 for ciprooxacin HCl and its related compounds. The sample solutions were freshly prepared and analyzed daily (Table 3). These experiments were repeated over a 1-week period to evaluate day-to-day variability and with a different analyst to assess intermediate precision of the developed method. As can be seen in Table 3, the RSD% values of the measurements were between 0.20 and 0.40% for ciprooxacin HCl whereas it ranged between 0.07 and 0.58% for the related compounds. The RSD% of assay results obtained in the intermediate precision study were not greater than 0.40%, conrming good precision of the proposed method between days and analysts.

Ciprooxacin HCl Impurity B Impurity C Impurity D Impurity E Chloro analogue

40.999 267.168 275.276 111.656 19.968 199.292

105.18 0.174 43.682 57.528 1.148 53.296

1.0000 0.9998 0.9974 0.9879 0.9987 0.9987

Table 3. Repeatability and intermediate precision data of HPLC assay of ciprooxacin HCl and its related compounds
Substance Theoretical concentration (lg mL)1) Repeatability Intra-day measured concentration Recovery (%) Ciprooxacin HCl Impurity B Impurity C Impurity D Impurity E Chloro analogue 500 1.0 1.0 1.0 1.0 1.0 100.47 102.55 102.31 101.77 101.45 102.38 RSD (%) 0.20 0.45 0.56 0.58 0.33 0.46 Intermediate precision Inter-day measured concentration Recovery (%) 100.56 99.22 99.28 99.70 99.95 99.41 RSD (%) 0.15 0.17 0.14 0.27 0.16 0.20 Dierent analysts measured concentration Recovery (%) 100.00 102.34 101.72 101.33 100.82 102.02 RSD (%) 0.40 0.08 0.08 0.17 0.16 0.07

Accuracy
Accuracy of the proposed method was established by recovery experiments. This study was employed by addition of known amounts of ciprooxacin HCl or its related compounds onto the tablet matrix (placebo powder). The resulting mixtures were analyzed as described for tablets. Results obtained from recovery studies are given in Table 4. The recovery experiments for ciprooxacin HCl, show mean recoveries of 100.18, 100.30 and 99.23% with RSD% values of 0.57, 0.18 and 0.41% at 80, 100 and 120% level, respectively. The recoveries were found Original

between 99.41 and 102.81% with RSD% values between 0.18 and 1.12% for the related compounds. High recovery results obtained from the proposed assay and impurities method for the analysis of ciprooxacin HCl lm-coated tablets indicate that this assay procedure can be used for quantitation and routine quality control analysis of this pharmaceutical dosage forms.

Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaected by small, but deliberate, variations in method parameters, and provides an indication of its reliability

during normal usage [23]. In the present study, an experimental design was planned for robustness testing varying some conditions, e.g. column temperature, pH of buffer in the mobile phase and age of columns (different batches of the same column). The results are shown in Table 5. It can be seen that, with every employed condition, there were no dramatic changes in the chromatographic parameters monitored (k, T, N) for ciprooxacin HCl, imp-C, imp-D and the chloro analogue. All parameters have been observed within the limits required for system suitability tests. Therefore, it can be concluded that the method is consistent in terms of temperature, pH of the buffer and column change (different batches of the same column).

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Table 4. Results obtained from recovery analysis of ciprooxacin and its related compounds at three concentration levels
Substance Added (lg mL)1) Found (lg mL)1) Recovery (%) RSD (%)

80% Level of the test concentration Ciprooxacin HCl 400.0 Impurity B 0.800 Impurity C 0.800 Impurity D 0.800 Impurity E 0.800 Chloro analogue 0.800 100% Level of the test concentration Ciprooxacin HCl 500.0 Impurity B 1.000 Impurity C 1.000 Impurity D 1.000 Impurity E 1.000 Chloro analogue 1.000 120% Level of the test concentration Ciprooxacin HCl 600.0 Impurity B 1.200 Impurity C 1.200 Impurity D 1.200 Impurity E 1.200 Chloro analogue 1.200

400.7 0.810 0.819 0.812 0.795 0.816 501.5 1.026 1.028 1.021 1.009 1.028 595.4 1.227 1.225 1.218 1.217 1.228

100.18 101.26 102.43 101.54 99.41 101.99 100.30 102.64 102.81 102.08 100.88 102.75 99.23 102.29 102.09 101.53 101.44 102.33

0.57 0.60 0.63 0.51 0.33 0.52 0.18 0.44 0.53 0.63 1.12 0.37 0.41 0.76 0.71 0.61 0.72 0.80

Table 5. Robustness evaluation of the developed HPLC method

Chromatographic Ciprooxacin changes HCl k A: Temperature 35 40a 45 T N Impurity C k T N Impurity D k T N Chloro analogue k T N

For heat and light (UV-visible) studies, the study period was 24 h whereas for acid and base hydrolysis, it was 72 h. The results obtained from forced degradation studies are summarized in Table 6. No appreciable degradation was observed for imp-D and chloro analogue in stressed samples (Cipro 500 mg lmcoated tablets) that were subjected to light, heat, acid and base hydrolytic conditions. Ciprooxacin was degraded into imp-C (ethylenediamine analogue) during base, thermal and photo-degradation conditions. This was conrmed with increase of % assay of this impurity in comparison to the initial situation (% assay at beginning). It was found that the content of imp-C after 72 h in 0.1 N NaOH rised from 0.027 to 0.040%. Earlier reports on the stability of ciprooxacin also supports that piperazine moiety of the molecule is sensitive to ring opening especially under photolytic conditions [2628]. In our case, exposure to dry heat, visible or UV light produced only little degradation to imp-C, showing that ciprooxacin in the lm-coated tablets was protected against heat and humidity.

4.673 1.372 8,477 2.242 1.296 4,240 4.604 0.785 9,451 8.491 1.001 11,251 4.051 1.306 8,562 1.899 1.161 8,019 4.043 0.730 8,861 7.208 1.024 10,863 3.516 1.251 8,689 1.659 1.202 5,727 3.610 0.791 8,539 6.250 1.039 11,108

Application of the Validated Method to Pharmaceutical Products

The applicability of the validated method was also tested by analyzing ciprooxacin HCl in pharmaceutical dosage forms. Assay results shown below represent the average of seven determinations. Analysis of Cipro 500 mg lm-coated tablets, which was claimed to contain 500 mg of the active substance, gave the mean assay values of 497.04 1.69 (SD) mg. Recovery and RSD% of the assay method were 99.41 and 0.34%, respectively. The differences between the amount claimed and those assayed were very low and the RSD value was less than 1%. The results of the quantitative analysis of lm-coated tablets which are summarized in Table 7, indicate that the proposed assay method can be used for quantitation and routine quality control analysis of ciprooxacin HCl in pharmaceutical dosage forms.

B: pH of the buer 2.9 2.609 1.186 7,542 1.595 1.184 8,454 3.399 0.818 8,861 6.014 1.032 11,625 4.051 1.306 8,562 1.899 1.161 8,019 4.043 0.730 8,861 7.208 1.024 10,863 3.0a 3.1 2.642 1.181 7,873 1.391 1.144 8,263 2.973 0.951 8,635 5.211 1.024 11,901 C: Column batch to batch variability 4.051 1.306 8,562 1.899 1.161 8,019 4.043 0.730 8,861 7.208 1.024 10,863 Column 1a Column 2 2.518 1.188 7,240 2.404 1.148 9,351 4.968 0.846 12,485 8.908 1.013 13,495
a

Optimum conditions

Specicity
Specicity can be described as the ability of the method to measure the analyte response in the presence of its potential impurities or other interferences such as excipients used in the formulations [23, 24]. Stress testing of the APIs is utilized for identication of the possible degradation products, and for validation of the stability indicating power of the analytical procedures used. High percentage recovery of ciprooxacin HCl observed with assay samples of pharmaceutical dosage forms, including recovery experiments, indicates that the proposed method was not affected by interferences from excipients used in formulations.

The specicity of the developed LC method for ciprooxacin was carried out in the presence of its impurities namely imp-B, imp-C, imp-D, imp-E and chloro analogue. Of these related compounds, imp-C and imp-E are possible degradation products of ciprooxacin whereas the remaining ones were reported as by-products of synthetic procedure. Stress studies were performed on ciprooxacin HCl lmcoated tablets to provide an indication of the stability indicating property and specicity of the proposed method. The stress conditions carried out for forced degradation study comprised acid hydrolysis (0.1 N methanolic HCl), basic hydrolysis (0.1 N NaOH), eective visible light, ultraviolet light (254 nm) and heat (60 C). Chromatographia Supplement Vol. 66, 2007

Conclusion
The validated stability-indicating HPLC method has proved to be simple, accuOriginal

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Table 6. Summary of forced degradation results

Stress condition Time (h) % Assay(w/w) of the impurities Imp-C Imp-D Chloro analogue Total impurities

Base hydrolysis 0.1 N NaOH Acid hydrolysis 0.1 N HCl Photodegradation Visible light

0 72 0 72 0 3 24 0 3 24 0 3 24

0.027 0.040 ND ND 0.0083 0.0088 0.0094 0.0083 0.0086 0.0093 0.0083 0.0086 0.0094

0.024 0.025 ND ND 0.016 0.016 0.015 0.016 0.016 0.014 0.016 0.019 0.014

0.038 0.036 0.013 0.013 0.038 0.038 0.038 0.038 0.038 0.037 0.038 0.038 0.038

0.089 0.100 0.013 0.013 0.062 0.063 0.062 0.062 0.063 0.060 0.062 0.066 0.060

UV light (254 nm)

Thermal degradation Dry heat (60 C)

Table 7. Results of the determination of ciprooxacin HCl in lm-coated tablets

Assay results Labelled claim (mg) Mean amount of found (mg) Number of samples SD of the amount found RSD% of the amount found Recovery from the tablets (%) 95% Condence interval 500.00 497.04 7 1.69 0.34 99.41 495.79498.30

can be used for the routine analysis of lm-coated tablets containing 500 mg of ciprooxacin hydrochloride and also check the purity and stability of the active substance in pharmaceutical dosage forms.

Acknowledgments
The authors greatly acknowledge Biofarma Pharmaceutical Industry Co. _stanbul, Turkey, for providing the Inc., I gift samples of ciprooxacin hydrochloride and its related compounds as well as Cipro 500 mg lm-coated tablet specimens and placebo powder mixture utilized throughout this study.

rate, precise, rapid and reliable. The proposed method provides a good resolution between ciprooxacin HCl and its potential impurities. Using this single procedure, it is possible to perform quantitative analysis of either ciprooxacin HCl or its related compounds in pharmaceutical dosage forms within 18 min. The developed method reported herein was validated by evaluation of the validation parameters as described in ICH guidelines. System suitability, specicity, linearity, LOD, LOQ values, precision, accuracy and robustness of the proposed technique were obtained during the validation studies. The developed method is also stability-indicating and

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