DEVELOPMENT AND VALIDATION OF A RAPID RP-HPLC METHOD FOR ESTIMATION OF SPARFLOXACIN IN TABLET DOSAGE FORM

Received on 27 September, 2013; received in revised form, 29 November, 2013; accepted, 14 January, 2014; published 01 February, 2014

DEVELOPMENT AND VALIDATION OF A RAPID RP-HPLC METHOD FOR ESTIMATION OF SPARFLOXACIN IN TABLET DOSAGE FORM

Ashok Kumar Bera ¹, Amit Kumar De ² and Biswajit Pal*³

Shri Ritam Vidyapith ¹, 293/1 Raja Rammohan Roy Road, Kolkata700041, West Bengal, India

R & D Division, Dey’s Medical Stores (Mfg) Ltd. ², 62 Bondel Road, Kolkata700019,West Bengal, India

Department of Chemistry, St. Paul’s C. M. College ³, 33/1 Raja Rammohan Roy Sarani, Kolkata 700009, West Bengal, India

ABSTRACT: The study describes development and subsequent validation of a reverse phase (RP) high-performance liquid chromatography (HPLC) method for the estimation of sparfloxacin (SPR) in tablet dosage form. The liquid chromatographic separation was achieved isocratically using a mobile phase composed of phosphate buffer (5.3 mM) and acetonitrile in the ratio of 60:40 (v/v) adjusted to pH 3.5 using dilute phosphoric acid. The analysis was carried out using X terra column (4.6 ´ 150 mm, 5µm, C₁₈) at a flow rate of 0.5 ml/min and the UV detection at 298 nm. The method was statistically validated for accuracy, precision, linearity, ruggedness, robustness and selectivity. The linearity of the proposed method was investigated in the range of 4 to 24 µg/ml (r² =0.999). Quantitative and recovery studies of the dosage form were also carried out and analyzed, the % RSD from recovery studies was found to be within the limit. Due to simplicity, rapidity and accuracy of the method we believe that the method will be useful for routine quality control analysis.

Sparfloxacin; RP-HPLC; Estimation; Validation

INTRODUCTION:Sparfloxacin is a long acting broad spectrum synthetic antibacterial agent belonging to the class Fluoroquinolone. It is used for the treatment bacterial infections like other fluoroquinolones. It is a DNA-gyrase and topoisomerase IV inhibitor used for the treatment of urinary tract and respiratory tract infections. It is also used for the treatment of bacterial infection in eyes ¹. The drug is found to be effective in community acquired pneumonia ², drug resistant tuberculosis ³, skin and soft tissue infection ⁴ and complicated urinary tract infections ⁵. 

The drug is found to be effective against a wide range of gram positive and gram negative bacterial, glucose non-fermentors and anaerobes, Legionella, Mycoplasma. Chlamydia, Mycobacterium species, Methicillin-resistant Staphylococcus aureus ⁶.

Chemically sparfloxacin is 5-amino-1-cyclopropyl-7-[(3R,5S)3,5-dimethylpiperazin-1-yl]-6,8-difluoro -4-oxo-quinoline-3-carboxylic acid as presented in Figure 1.

FIGURE 1: CHEMICAL STRUCTURE OF SPARFLOXACIN

The molecule has got poor aqueous solubility resulting poor G.I. tract absorption. Its biological half-life is 15 to 22 hours in healthy volunteers after oral administration ⁷. The molecule gets well distributed throughout the body with a higher concentration in sinus mucosa, bronchial mucosa, epithelial lining fluid, and alveolar macrophages ⁸ and  is the drug of choice for treating the lower respiratory tract infection.

Extensive literature survey presented a fewer number of suitable analytical method for the estimation of Sparfloxacin from oral dosage forms and ophthalmic preparations. Only few spectro-photometric methods ^{9, 10} and chromatographic ^11-13 methods have been reported. A rapid, precise, sensitive chromatographic method for the estimation of Sparfoloxacin from oral dosage forms is therefore essential. Reported methods are somewhat tedious and time consuming ^9-13.

In our current study, a validated rapid analytical method for the estimation of Sparfloxacin has been developed using reverse phase chromatographic technique.

EXPERIMENTAL:

Reagents and chemical: Potassium di-hydrogen phosphate and dipotassium hydrogen phosphate of AR grade, phosphoric acid and acetonitrile of HPLC grade were purchased from Merck Ltd., Mumbai. Reference standard of sparfloxacin was procured from Central Drug laboratory, Kolkata. Tablet of brand Novospar tablet (Zydus Cadila) having of label claim of sparfloxacin 200mg was used. Water of HPLC grade was obtained from Aurium 611 UV water purifier of Sartorius, Germany.

Equipment: Chromatographic separation was performed on Waters Alliance e2695 HPLC module having 2489 dual Lambda UV absorbance detector. Empower 3 software was applied for data collecting and processing.

HPLC conditions: A C₁₈ X terra column (150 mm × 4.6 mm, 5 µm) was used for separation. Mobile phase consisting of a mixture of 60 volumes of 5.3 mM phosphate buffer solution adjusted to pH 3.5 ± 0.1 with orthophosphoric acid and 40 volumes of accetonitrile was pumped at 0.5 ml /min.

The mobile phase was filtered through a 0.45 µm membrane filter and degassed. The eluents were monitored at 298 nm. The injection volumes of sample and standard were 10 µl.

Standard preparation: Standard stock solution of 0.5 mg/ml of sparfloxacin was prepared in 10 ml HPLC grade water by addition of 1 drop of conc. HCl followed by sonication for 15 min and the final volume was made by the mobile phase. The final working standard solution of sparfloxacin had a final concentration of 0.02 mg/ml and was prepared by appropriate dilution with mobile phase from the stock solution. The content of standard solution was filtered through 0.45 µm syringe filter.

Sample preparation: Twenty tablets were weighed and crushed into fine powder. An accurately weighed quantity of powder equivalent to 10.0 mg of sparfloxacin was transferred into a 25 ml volumetric flask. About 10 ml of water and 1 drop of conc. HCl was added to the volumetric flask and sonicated for 15 minutes and volume was made up to the mark by mobile phase. This solution was filtered through Whatman No. 1 and further diluted to 0.016 mg/ml of sparfloxacin (theoretical value) in mobile phase. The content of sample solutions was filtered through 0.45 µm syringe filter.

Method validation: The developed method was validated as per ICH guidelines in terms of parameters like specificity, precision, accuracy, linearity and range, LOD, LOQ, ruggedness, robustness etc.^{14, 15}.

System suitability: System suitability tests were carried out on freshly prepared solution of reference standards of sparfloxacin to check various parameters like retention time, tailing factor, theoretical plate etc. The results were shown in Table 1.

TABLE 1: SAMPLE SUITABILITY PARAMETER

Linearity and range: The linearity was studied to determine the range over which analyte response in a linear function of concentration. The study was performed by preparing standard solutions of the sparfloxacin at five different concentrations and analyzes were performed in triplicate. Calibration curve was constructed by plotting average peak area against concentration and regression equation was computed. The results were shown in Table 2. The linearity curve of sparfloxacin was represented in Figure 2.

TABLE 2: LINEARITY PARAMETERS

FIGURE 2: SOLUTION CONCENTRATION VERSUS PEAK AREA LINEARITY CURVE

Precession: The precession of an analytical method in the closeness of replicate results obtained from analysis of the same homogeneous sample. The intra-day precision of the proposed method was determined by making six replicate injections of the standard solution on the same day. Inter-day precision was evaluated by analysing the same sample in the same way on different days. The percentage RSD with respect to the peak area, peak retention time and amount were calculated for each case and presented in Table 3. The lower values of %RSD prove that the method is precise ¹⁴.

Assay: 10 µl standard and sample solutions were injected on HPLC system using optimized mobile phase and other chromatographic conditions. Chromatograms of standard solutions (six replicates) and sample solutions (two replicates) were recorded. Typical chromatogram of sparfloxacin was presented in Figure 3. The concentration of sparfloxacin in the tablet formulation was calculated by comparing area of the sample with that of the standard. The percentage assay of the drug was calculated and presented in Table 4.

FIGURE 3: REPRESENTATIVE CHROMATOGRAM FOR SPR (RETENTION TIME = 2.528)

Accuracy: The accuracy of an analytical method is the closeness of results obtained by that method to the true value for the sample. To evaluate the accuracy of the proposed method, recovery tests were carried out. Recovery tests were performed by adding known amounts of standard solutions (90%, 110% and 120%) to the pre-analysed sample followed by analysis using the proposed method. The recovery studies were performed in triplicate and percentage recovery and standard deviation of the percentage recovery were calculated and presented in Table 5. The lower values of %RSD of assay indicate the method is accurate and there is no interference from excipients ^{14, 15}.

TABLE 3: PRECISION PARAMETERS

TABLE 4: ASSAY PARAMETERS

TABLE 5: ACCURACY PARAMETERS (RECOVERY STUDIES)

Limit of detection and limit of quantification: The limit of detection (LOD)  in the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3) and the limit of quantitation (LOQ) is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10). The LOD and LOQ of the developed method were determined by injecting progressively low concentration of the standard solution using the developed RP-HPLC method. The results were shown in Table 1.

Ruggedness and robustness: The ruggedness of the proposed method was determined by carrying out the experiment on different instruments like Merk Hitachi HPLC La Chrome pump L-7100 with Merk Hitachi UV La Chrome detector L-7400, Waters HPLC 600 pumps by different operators using different columns of similar type ¹⁴.

Robustness of the method was determined by making slight changes in the Chromatographic conditions, such as change in mobile phase, flow rate and column temperature. It was observed that there were no marked changes in the chromatograms and did not affect the recovery of the drug which indicated that the proposed method is rugged and robust.

The robustness limit for mobile phase variation, flow rate variation and temperature variation are well within the limit, which shows that the method is having good system suitability and precision under given set of conditions and were within the acceptance criteria of not more than 2%.

RESULTS AND DISCUSSION: In order to validate our proposed method, parameters such as detection wavelength, ideal mobile phase, optimum pH and concentrations of the standard solutions were exhaustively studied. Of several solvents and solvent mixtures investigated, the mobile phase consisting of phosphate buffer (5.3 mμ) and acetonitrile in the ratio of 60:40 (v/v) adjusted to pH 3.5 was found to furnish sharp, well defined peaks with very good symmetry (Fig. 2) at wavelength 298 nm. The proposed method is simple and do not involve laborious time consuming sample preparation.

The proposed method is a cost effective or economical method because a flow rate of 0.5 ml /min was selected after preliminary test with retention time 2.528. The method was statistically validated as per ICH guideline. The analytical procedure was found to be linear in the concentration range 4 to 32µg/ml with regression factor 0.999. The percent RSD with respect to peak area, peak retention time and amount for sample solution were 0.06, 0.09 and 0.12 respectively and the values were 853514, 2.528 and 199.98mg/Tab respectively for intraday precision and 853465, 2.525 and 199.96mg/Tab respectively for interday precision (Table 3), indicating precision of the method. The recovery values obtained were between 99.55 % and 100.42% (Table 5), confirming accuracy of the proposed method.

The LOD and LOQ were found to be 0.5317µg/ml and 1.6111µg/ml respectively. The method was satisfactory with respect to ruggedness and robustness also.

CONCLUSION: The proposed HPLC method enables quantitative determination of sparfloxacin. The proposed method was found to be easy in preparation of sample, fast, precise, accurate, sensitive, efficient and economical and can be used in routine analysis in quality control laboratories.

ACKNOWLEDGMENTS: The authors gratefully acknowledge Dey’s Medical, Kolkata for providing necessary facilities for carrying out this study.

REFERENCES:

1. Nesseem DI: Ophthalmic delivery of sparfloxacin from in situ gel formulation for treatment of experimentally induced bacterial keratitis. Drug Test Anal 2011; 3:106 -115.
2. Aubief M, Lode H, Gialdroni-Grassi G, Huchon G, Hosie J, Legakis N,  Regamey C, Segev S, Vester R, Wijnands WJ, Tolstuchow N: Sparfloxacin for the treatment of community-acquired pneumonia: a pooled data analysis of two studies. J Antimicrobial Chemotherapy 1996; 37:73 - 82.
3. Lubasch A, Erbes R, Mauch H, Lode H: Sparfloxacin in the treatment of drug resistant tuberculosis or intolerance of the first line therapy. Eur Respir J 2001; 17:641- 646.
4. Iakovlev VP, Krutikov MG, Blatun LA, Izotova GN, Grishina IA: Clinical and laboratory efficacy of the novel difluoroquinolone sparflo (sparfloxacin) in therapy of skin and soft tissue infections. Antibiot Khimioter 2000; 45:37- 40.
5. Dereviako II, Lavrinova LN, Nefedova LA: Use of sparfloxacin (sparflo) in treating complicated urologic infections. Urologia  2002; 2:27-30.
6. Shimada J, Nogita T, Ishifashi Y:Clinical pharmacokinetics of sparfloxacin. Clin Pharmacokinet 1993; 25:358–69.
7. Bhar MK, Khargharia S, Chakraborty AK, Mandal TK: Disposition kinetics of sparfloxacin in healthy, hepatopathic, and nephropathic conditions in chicken after single intravenous administration.  Indian J  Pharmacol 2009; 41:106-109.
8. Wise R, Honeybourne D: A review of the penetration of sparfloxacin into the lower respiratory tract and sinuses. J Antimicrob Chemother 1996; 37: 57 - 63.
9. Shah J, Jan MR, Khan I, Khan MN: Quantification of sparfloxacin in pharmaceutical dosages and biological samples. Pak J Pharm Sci 2012; 25: 823 - 829.
10. Sharma MC, Sharma S: UV-densitometric determination of sparfloxacin and its application to the assay in pharmaceutical dosage forms. J Optoelectronics and Biomedical Materials 2010; 2:191- 195.
11. Noh K, Kwon KI, Jeong TC, Kang W: Quantitative determination of sparfloxacin in rat plasma by liquid chromatography/tandem mass spectrometry. Biomed Chromatogr 2010; 24 :1199 -1202.
12. Gupta H, Aqil M, Khar RK, Ali A, Sharma A, Chander P: Development and validation of a stability-indicating RP-UPLC method for the quantitative analysis of sparfloxacin. J Chromatogra Sci 2010; 48:1- 6.
13. Srinivas N, Narasu L, Shankar BP and Mullangi R: Development and validation of HPLC method for simultaneous quantitation of gatifloxacin, sparfloxacin andmoxifloxacin using levofloxacin as internal standard in human plasma: application to a clinical pharmacokinetic study. Biomed Chromatogr 2008; 22: 1288 - 1295.
14. United States Pharmacopeia. USP Convention,  Rockville, MD,  2008; p 1225
15. ICH (Q2, R1).Note for guidance on validation of analytical methods: Definitions and terminology. International conference on harmonization, 1994; 9-13.
    

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    How to cite this article:

    Bera AK, De AK and Pal B: Development and validation of a rapid RP-HPLC method for estimation of Sparfloxacin in tablet dosage form. Int J Pharm Sci Res 2014; 5(2): 563-67.doi: 10.13040/IJPSR.0975-8232.5(2).563-67

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