INTRODUCTION:

Prulifloxacin fig.1(6-Fluoro-1-methyl- 7-(4-(5-methyl-2-oxo-1,3-dioxelen-4-yl)methyl-1- piperazinyl)-4-oxo-4H-(1,3)thiazeto(3,2- a)quinoline-3-carboxylic acid) is a fluoroquinolone antibiotic. It is a prodrug of Ulifloxacin. Prulifloxacin belongs to fourth-generation fluroquinolones and has extensive Gram-negative coverage, good Gram-positive coverage and also possesses activity against anaerobes¹. It is mainly used in the treatment of bronchitis exacerbation and lower urinary tract infection². It shows its antibacterial activity by inhibiting DNA gyrase thus preventing DNA replication and synthesis^{3, 4}.

Various methods have been reported in literature for the analysis of prulifloxacin in biological fluids including spectrofluorimetric^5,6, capillary electrophoresis⁷ and LCMS⁸. However to our knowledge, no article related to high performance liquid chromatography (HPLC) determination of prulifloxacin in bulk and pharmaceutical dosage forms has reported in literature.

A simple analytical method is required that can quantitatively estimate prulifloxacin in the bulk and its dosage form.

This paper describes the development and validation of a stability indicating RP-HPLC method for the assay of prulifloxacin as a bulk drug and in its pharmaceutical dosage forms.

EXPERIMENTAL:

Materials

Prulifloxacin was obtained as a gift sample from Hetero durgs, Hyderabad, India. Prulifloxacin tablets were procured from local pharmacy. All the reagents were of analytical grade. Double distilled water was used throughout the experiment.

HPLC instrumentation

HPLC model schimadzu LC20-AD HPLC system with Rheodyne universal injector 7725 and LC20-AD UV-Visible detector, and auto injector (20 μL).

Chromatographic conditions:

The chromatographic studies were performed using 250 mm x 4.6mm Inertsil ODS-3v,C18 column with 5μm particle size at ambient temperature and eluted with mobile phase consisting of 0.2% acetic acid in water, acetonitrile and methanol in the ratio (7:2:1 v/v), at the flow rate of 1ml/min. The run time was 24 min. The mobile phase was filtered through 0.45μm nylon filter and degassed in ultrasonicator prior to use. An absorption maximum of pure drug was scanned over the range 200-400nm wavelength by using ELICO SL-196 UV-Visible spectrophotometer with a pair of 10mm matched quartz cells. Observations were made with injection volume of 20μl and at a wavelength of 273nm.

Fig no. 1: Chemical structure of Prulifloxacin

Preparation of standard solution

Accurately weighed quantity of Prulifloxacin (10 mg) was dissolved in diluent (0.1% acetic acid in water: acetonitrile (10:90)) volume was made up to 100 mL with mobile phase (100 μg mL^-1).

Preparation of sample solution

Twenty tablets were weighed and finely powdered. An accurately weighed quantity of tablet powder equivalent to 10 mg of prulifloxacin was shaken with mobile phase for 5-10 min and volume adjusted to 100 mL with mobile phase. The solution was filtered. The filtrate was further diluted with mobile phase to get concentration of prulifloxacin equivalent to about 30 μg mL^-1.

Assay of Prulifloxacin tablets:

The developed method was applied to the assay of Prulifloxacin tablets. The content was calculated as an average of 6 determinations and experimental results were given in table 1. The results were very close to the labeled value of commercial tablets. The Representative model chromatogram of Prulifloxacin sample in a tablet was shown in fig 2.

Table no 1: Assay results of Prulifloxacin formulations

Formulation	Labeled amlunt (mg)	Amount found (mg)	% RSD*
Prunox	600	593	98.8 ± 0.54

*% RSD of 6 determinations

Fig. no. 2

RESULTS AND DISCUSSION:

Validation study:

Specificity:

Specificity of an analytical method is its ability to measure accurately and specifically the analyte of interest without interference from placebo and degradation products. The specificity of the method was established by injecting mobile phase and placebo solution in triplicate and recording the chromatograms. It was observed that there was no interference from the placebo with the analyte peak.

System suitability:

System suitability studies were conducted by taking 10 readings of standard solution at 100% of target concentration. The %RSD values less than the 2% for the retention times and peak areas of ten replicate injections indicate that the system is acceptable.

Method precision:

The repeatability of the instrumental system was evaluated with this parameter. Six sample solutions were prepared individually and injected each solution. The standard deviation and relative standard deviation were calculated for six injections. The % RSD values less than 2 indicate that the method was precise. Results are indicated in table no 2.

Table no 2: Precision of proposed method

CONCENTRATION OF PRULIFLOXACIN	OBSERVED CONCENTRATION OF PRULIFLOXACIN
	INTRA- DAY		INTER- DAY
	MEAN (n=3)	CV (%)	MEAN (n=3)	CV (%)
10	10.02	0.906	9.98	0.758
20	20.01	0.284	20.07	0.612
30	30.01	0.201	30.00	0.222
40	39.98	0.180	40.05	0.240

Intermediate precision:

Intermediate precision was performed by injecting the drug substance several times over a period of two days (initial, 6hrs, 12hrs, 18hrs, 24hrs, 30hrs, 36hrs, and 42hrs) by three different analysts and different solutions. The relative standard deviation was calculated. The % RSD values less than 2 indicate that the method was precise.

Accuracy (Recovery studies):

Accuracy of a method is defined as the closeness of a measured value to the true `value. The recovery studies were done at 50%, 100%, and 150% of the target level in the tablet in triplicate each in the presence of placebo. The recovery was calculated with respect to the standard deviation. The results were tabulated in table 3. The mean % recovery of Prulifloxacin at each level was not less than 95% and not more than 105%. These results indicate that the method has an acceptable level of accuracy.

Table no 3: Results of Accuracy (Recovery studies)

AMOUNT OF DRUG ADDED(µg/ml)	RECOVERY OF DRUG SOLUTION
AMOUNT OF DRUG ADDED(µg/ml)	AMOUNT FOUND(µg/ml)	MEAN% RECOVERY
10	10.03	100.01
20	20.09	100.45
30	29.48	98.27

Linearity:

Linearity is the ability of the method to respond proportionally to the changes in the concentration of the analyte in a sample. Linearity studies were conducted by using series of standard solutions of Prulifloxacin at concentration levels from 25- 150% of the target concentration (25%, 50%, 75%, 100%, 125% and 150%). This linearity studies are repeated for three times with different stock solutions. The calibration curve was obtained by plotting the peak area Vs concentration of the standard solution. The correlation coefficient value greater than 0.999 indicate that the method was linear from 25-150% of the target concentration.

Limit of detection (LOD):

Limit of detection is the smallest concentration that can be detected reliably. LOD is determined based on signal to noise ratio (3:1) and it was found to be 0.5μg/ml.

Limit of quantitation (LOQ):

Limit of quantitation of the analyte that can be determined accurately. LOQ is also determined based on signal to noise ratio (10:1) and it was found to be 1.5μg/ml.

CONCLUSION:

The proposed high performance liquid chromatographic method has been evaluated for linearity, precision, accuracy, specificity and proved to be convenient and effective for the estimation of Prulifloxacin in tablet dosage forms.

ACKNOWLEDGMENT:

The authors are grateful to management of Vijaya College of Pharmacy, Hyderabad for providing laboratory facilities.

REFERENCE:

1. G. Giannarini, C. Tascini, C. Selli, Future Microbiology, 2009, Vol 4, pp13-24.

2. Hooper, D. Q., in: Mandell, G. L., Bennett J. E., Dolin R. M., Douglas and Bennett’s principles and practice of infectious diseases, Philadelphia, Churchil Livingstone, 2000, pp. 656-662.

3. Hooper, D. C., In: Wolfson, J. S., Quinolone Antimicrobial Agents, American society for microbiology, Washington, 1993, pp 53-57.

4. D. C.Hooper, Drugs, 1999, Vol 58, pp6.

5. Fengshan Yu, Lin Li , Fang Chen, Anal. Chimi. Acta, 2008, Vol 610, pp257-262.

6. Fengshan Yu, Yongwei Gao, Man Cui, Fang Chen , Yanbin Ding, Analytical Letters, 2009, Vol 41, pp2933-2943.

7. L. Zhang, J. Wen, Y. Pan, Z. Li, G. Fan, Y. Wu, J. Chrom. B, 2008, Vol 287, pp172-177.

8. Guo L, Qi M, Jin X, Wang P, Zhao H., Determination of the active metabolite of prulifloxacin in human plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Mar 7;832(2):280-5. Epub 2006 Feb 20