INTRODUCTION :

Moxifloxacin Hydrochloride (MOX) is chemically 7-[(4aS, 7aS)-octahydro-1H-pyrrolo [3, 4-b] pyridin-6-yl]-1 cyclopropyl-6-fluoro-8-methoxy-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid. It is a synthetic antibacterial agent used for the treatment of sinus and lung infections such as sinusitis, pneumonia, and secondary infections in chronic bronchitis and also for the treatment of bacterial conjunctivitis. Ketorolac Tromethamine (KT) is chemically 5-benzoyl-2, 3-dihydro-1H-pyrrolizine-1-carboxylic acid. It is a nonsteroidal anti-inflammatory drug (NSAID) used to relieve the eye pain and inflammation after cataract extraction and keratorefractive surgery. Few methods such as spectrophotometric method ^1-3,8, HPLC ^5-13, method and HPTLC ^4,14, method have been reported for individual drugs or in combination with other drugs in formulation. So far no simultaneous method has been reported for estimation of MOX and KT. The aim of this study was to develop a precise, specific, accurate and sensitive method for simultaneous determination of MOX and KT combination by RP-HPLC method.

MATERIALS AND METHODS:

MOX and KT were obtained from Zydus Cadila Healthcare Ltd, Moraiya, Ahmadabad, India. Acetonitrile HPLC grade of Rankem, OPA HPLC grade of Merck, Ammonium Acetate AR grade of Merk chemicals were used for the study. A Shimadzu Isocratic system was used coupled with UV detector and Rheodyne injector with 20 μl fixed loop. Chromatographic analysis was performed using Spinchrome software on a Hypersil C₁₈ column (250x4.5mm, 5μ) at ambient temperature. The mobile phase consisting of Acetonitrile: Buffer (pH-4.0) (60:40) was pumped at a flow rate 1.0 ml per min; the detection was monitored at 294 nm.

Preparation of stock solution of Moxifloxacin Hydrochloride and Ketorolac Tromethamine:

Standard stock solutions were prepared by dissolving 50mg of MOX and 50 mg of KT in the 500 ml flask and diluted to 500 ml with the mobile Phase. In case of MOX and KT varying amounts of (1.0, 3.0, 5.0, 7.0 and 9.0) of the above stock solution of mixture of MOX and KT (100μg/ml) was taken in five different 10 ml volumetric flasks and the volume was made up to the mark with the mobile phase. An aliquot of 20μl of the solution from each flask was injected into the system.

Chromatographic Conditions: Calibration curve:

An aliquot of 20μl of the solution from each flask was injected two times. Calibration curves were constructed by plotting mean peak areas against the corresponding drug concentrations. MOX and KT were found to be linear in the range of 10-90 μg/ml and 10-90 μg/ml with coefficient of correlation (r²) 0.998 and 0.998 for MOX and KT, respectively.

Determination of MOX and KT in their combined dosage forms:

A quantity equivalent to 50μg/ml (1 ml) of MOX and KT was transferred to a 100 ml volumetric flask and 30 ml of mobile phase was added. The flask was shaken for 15 min and then contents were diluted to 100 ml and filtered through Whatman No 41filter paper. Results of the triplicate analysis are given in Table 1. The method was validated for statistical parameters i.e. precision, accuracy, specificity, linearity and ruggedness criteria. Results of the method validation experiments are given in Table 2. The precision of the method was determined by knowing percentage RSD of means of three replicate solutions of all the three independent samples.

TABLE 1: ASSAY RESULTS OF COMBINED DOSAGE FORM

Labelled amount (mg/ml)

Amount obtained (mg/tablet ± RSD*)

% Assay*

MOX

4.93 ± 0.20

5.01 ± 0.37

98.67

100.39

*Denotes average of five determinations. MOX and KT denote, Moxifloxacin and Ketorolac, respectively.

TABLE 2: VALIDATION AND SYSTEM SUITABILITY PARAMETERS

Parameter	Moxifloxacin	Ketorolac
Linearity Range (μg/ml)	10 – 90	10 – 90
Correlation Coefficient (r2) ± S.D*	0.998 ± 0.1	0.998 ± 0.5
Retention time (min.) ± S.D*	3.54 ± 0.07	5.62 ± 0.01
Resolution	12.065
Tailing factor	1.4	1.3
Theoretical plates	8574	13639
Limit of detection (μg/ml)	0.54	0.73
Limit of quantification (μg/ml)	1.07	3.24
Precision (RSD, %) Intraday (n=5)	0.86	1.57
Repeatability (RSD, %) (n=5)	1.17	1.03

* Mean of five determinations (n=5).

TABLE 3: RECOVERY STUDIES OF MOX AND KT IN COMBINED DOSAGE FORM

Moxifloxacin			Ketorolac
% added	% recovered ± % RSD*	% recovery	% added	% recovered ± % RSD*	% recovery
80	99.51 ± 1.28	99.51	80	80.01 ± 0.99	100.01
100	100.83 ± 0.53	100.83	100	99.45 ± 1.24	99.45
120	118.89± 0.95	99.08	120	120.30 ± 0.94	100.25

*Mean of three determinations (n=3). MOX and KT denote, Moxifloxacin and Ketorolac respectively.

Figure 1 Chromatogram of Moxifloxacin (MOX) and Ketorolac (KT).

Precision:

The intra day precision study of MOX and KT was carried out by estimating the corresponding responses five times on the same day and the results are reported in terms of relative standard deviation (RSD, Table 2).

Accuracy:

The accuracy of method is determined by adding known amount of standard to that of sample (above and below the normal level) at 3 different levels to cover both above and below (80% to 120%) the normal levels expected in the sample.

Specificity:

The specificity of the RP-HPLC method was determined by complete separation of MOX and KT as shown in Fig No. 1 with parameters like retention time (tR), resolution (Rs) and tailing factor (T). Here tailing factor for peaks of MOX and KT was less than 1.5% and resolution was satisfactory. The peaks obtained for MOX and KT were sharp and have clear base line separation.

Detection limit and quantification limit:

A calibration curve was prepared by using concentration in the expected detection limit range of 0.5- 0.7 μg/ml for MOX and KT. The standard deviation of y-intercepts of regression lines were determined and kept in the following equation for the determination of detection limit and quantification limit. Detection limit = 3.3 σ/s; quantification limit = 10 σ /s; Where σ is the standard deviation of y intercepts of regression lines and s is the slope of the calibration curve.

Robustness:

Robustness of the method was studied by deliberate variations of the analytical parameters such as flow rate (1.0± 0.2 ml/min), pH of the mobile phase (pH 4 ± 2).

RESULT AND DISCUSSION:

Optimization of the mobile phase was performed based on resolution, asymmetric factor and peak area obtained for both MOX and KT. The mobile phase Acetonitrile: Buffer (60:40) was found to be satisfactory and gave two symmetric and well resolved peaks for MOX and KT. The resolution between MOX and KT was found to be 12.06 which indicate good separation of both the compounds. The retention time for MOX and KT were 3.42 min and 5.62 min, respectively (Fig. no 1). The asymmetric factors for MOX and KT were 1.4 and 1.3, respectively. The calibration curve for MOX was obtained by plotting the peak area of MOX versus the concentrations of MOX over the range of 10-90 μg/ml, and it was found to be linear with r² = 0.998. Similarly, the calibration curve for KT was obtained over the range of 10-90 μg/ml and was found to be linear with r² = 0.998. The data of regression analysis of the calibration curves and the validation parameters are summarized in Table 2. The quantitation limit for MOX and KT were 1.07 and 3.22 μg/ml respectively. The recoveries of MOX and KT were found to be in the range of 99.08-100.83% and 99.45-100.25%, respectively. The system suitability test parameters are shown in Table 2. The chromatographic method was applied to the determination of MOX and KT in their combined dosage forms (Tablet formulation). The result for MOX and KT were comparable with the corresponding labelled amounts (Table 1). Proposed study describes a new RP-HPLC method for estimation of MOX and KT combination in mixture using simple mobile phase. The method gives good resolution between both the compounds with a short analysis time. The method was validated and found to be simple, sensitive, accurate and precise. Percentage recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of MOX and KT in their combined dosage form.

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Received on 04.04.2012 Modified on 10.05.2012

Asian J. Research Chem. 5(5): May 2012; Page 697-699