INTRODUCTION:

Chemically Ofloxacin (Oflox) is (±) -9-fluro-3-methyl-10-(4-methyl-1-piperazinyl)-oxa-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, fluroquinolone¹ and Ornidazole (Orni) is [α-(chloromethyl-2-methyl-5-nitroimidazole]ethanol)². Literature reveals that few Spectrophotometric^3,4, Capillary Zone Electrophoresis⁵, HPLC ^6,7,8,9, LCMS¹⁰and HPTLC¹¹ methods are reported for estimation of Ofloxacin and Ornidazole alone and in combination. No method was found for simultaneous estimation of Oflox, Orni and its isomer. So, in the present investigation an attempt has been made to develop accurate and precise RP-HPLC method for simultaneous estimation of Oflox and Orni with its isomer in combined dosage form.

MATERIAL AND METHODS:

Ofloxacin (Oflox) and Ornidazole (Orni) bulk powders were kindly gifted by Vapi Care Pharma Pvt. ltd, Vapi. HPLC grade water, acetonitrile and methanol were procured from Loba Chemie Pvt. Ltd., Mumbai.

Instrumentation:

Analysis of all the samples were performed using JASCO PU 1580 HPLC system consisted of a intelligent pump, a 1575 intelligent UV-Visible detector with precision loop injector (Rheodyne, 20µl).

The data was processed using Borwin 1.27 software. All the samples were filtered through whatman filter paper no. 41 and degassed by sonication for 15min.

Chromatographic condition:

RP-HPLC method was developed on JASCO intersil C18, 4.6 (i.d.) x 250 mm, 10µm column. A mobile phase containing acetonitrile: methanol: 0.025M phosphate buffer (pH 3.0) (30:10:60 v/v/v) was selected because the peaks of Oflox (Rt = 4.04min), Orni and its isomer (Rt = 5.82, 6.77 min) were ideally resolved as shown in Figure 1. Wavelength was selected at 318 nm by scanning standard solution of both the drugs over 200-400 nm wavelength. Measurements were made at the flow rate of 1.0 ml/min with injection volume 20µl.

Preparation of standard stock solution:

Standard stock solution containing Oflox and Orni were prepared individually by dissolving 100 mg of Oflox and 250 mg of Orni in methanol. The final dilution of both the solution were made up to 100 ml with methanol to get stock solution containing 1000 µg/ml of Oflox and 2500 µg/ml of Orni.

Calibration Curve:

An aliquot portion of standard stock solution of Oflox and Orni were further diluted with mobile phase to the series of concentration ranging from 0 – 40 µg/ml for Oflox and 0 -100 µg/ml for Orni. The mobile phase was allowed to equilibrate with stationary phase until the steady baseline was obtained. Then each dilution of both the drug was injected and the peak area was recorded. The calibration curve was constructed by plotting concentration of the drug against average peak area and regression equation was computed.

Figure 1. Chromatogram of Oflox, Orni and its isomer with Rt 4.04, 5.82 and 6.77 min respectively

.

Validation of method:

The developed method was validated in terms of accuracy, precision, linearity and range, ruggedness study.

Sample preparation:

Twenty tablets (OFLOX OZ, Cipla Ltd., Jaipur) were weighed and finely powdered. An accurately weighed quantity of powder equivalent to 100 mg of Oflox (equivalent to 250 mg of Orni) was taken in 100 ml of volumetric flask and dissolved in methanol. Volume was made up to the mark by methanol. The solution was sonicated for 15 min. and filtered through Whatman filter paper no. 41. Further dilutions were made by mobile phase.

RESULTS AND DISCUSSION:

To develop a precise, accurate and suitable RP-HPLC method for the simultaneous estimation of Oflox and Orni, different mobile phase were tried and the proposed chromatographic conditions were found to be appropriate for the quantitative determination. The results obtained by the assay of marketed formulation are summarized in table no. 1. System suitability testes were carried as per USP and parameters are summarized in table no. 2.

Table 1. Results of Oflox and Orni in marketed formulation

Drug Name	% Amount Found	% RSD
Oflox	100.23	0.24
Orni	99.61	0.35

Table 2. System Suitability Parameters

Parameters	Oflox	Orni I	Orni II
Asymmetry	0.853	0.679	0.912
Retention time (min)	4.04	5.82	6.77
Capacity Factor	2.464	3.796	4.838
Selectivity Resolution	1.53 10.06	1.27 4.57

Method Validation:

Accuracy:

The proposed HPLC method ascertained on the basis of recovery study performed by the standard addition method at 80%, 100% and 120% known amount of standard Oflox and Orni were added to preanalysed samples and were subjected to the propose HPLC method. Results of the recovery studies are shown in table no. 3.

Table 3. Recovery data

Drug Name	% Recovery	% RSD
Oflox	100.20	0.43
Orni	100.93	0.81

Precision:

Precision of an analytical method is expressed as S.D. and R.S.D. of the series of the measurements. It was ascertained by replicate estimation of marketed formulation (six times). The % R.S.D was found to be less than 2 % which proves that method is precise.

Linearity and Range:

According to ICH guidelines, five concentration levels were prepared in specified range of 80 to120% of the target concentration and peak areas were determined. The regression for Oflox and Orni was found to be Y = 0.6165X and Y = 0.4314X with coefficient of correlation (R²) 0.9994 and 0.9913 respectively.

Ruggedness:

Ruggedness was ascertained by carrying out the analysis for interday variation, intraday variation and different analyst. The results are summarized in table no 4.

Table 4. Ruggedness parameters

Parameters	Label Claim ± SD
Drug Name	Oflox	Orni
Interday Variation	100.28 ± 0.31	100.11 ± 0.61
Intraday Variation	100.12 ± 0.46	99.98 ± 0.65
Different Analyst	100.20 ± 0.40	100.15 ± 0.77

CONCLUSION:

The proposed method is simple, sensitive and reproducible and hence can be used in routine simultaneous estimation of Oflox, Orni and its isomer in bulk as well as in pharmaceutical preparation. Statistical analysis of the results has been carried out revealing high accuracy and precision. The RSD for all the parameters were found to be less than one, which indicates the validity of the method.

ACKNOWLEDGEMENT:

The authors are thankful to Vapi Care Pharma Pvt. Ltd. for providing the gift samples of Ofloxacin and Ornidazole.

REFERENCES:

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Received on 09.03.2010 Modified on 02.04.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 666-668