Int J Pharm Pharm Sci, Vol 6, Issue 9, 552-555Original Article

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE ESTIMATION OF SITAFLOXACIN IN BULK AND PHARMACEUTICAL DOSAGE FORMS

NAGA RAJU POTNURI*1, DEVALA RAO G2, RAJENDRA PRASAD Y3

1Department of Pharmaceutical Analysis, Joginpally B. R. Pharmacy College, Yenkapally, Moinabad, R. R. Dist, A. P, India, 2Department of Pharmaceutical Analysis, K. V. S. R Siddhartha College of Pharmaceutical Sciences, Vijayawada, A. P, India, 3Department of Pharmaceutical Chemistry, University Colleges of Pharmaceutical Sciences, Andhra University, Vishakhapatnam, A. P, India
Email: nagaraju_potnuri@yahoo.co.in

Received: 16 Jun 2014 Revised and Accepted: 04 Sep 2014

ABSTRACT

Objective: A simple, specific, and precise reversed phase high performance liquid chromatography method was developed and validated as per the ICH guidelines for the determination of Sitafloxacin in bulk and tablet dosage forms.

Methods: The quantitation was carried out by using Symmetry C18 ((5 µm, 100X4.6 mm) column at 300c with Water: Acetonitrile in ratio of 70:30 % V/V as mobile phase. The flow rate is 0.9 mL/min and the estimation was carried out by using PDA detector at 300 nm.

Results: The retention time of Sitafloxacin was 2.198 minutes. The linearity was observed from 5-25 μg/mL with correlation coefficient 0.9999. The LOD and LOQ were found to be 0.429μg/mL and 1.415μg/mL respectively.

Conclusion: The Statistics data for the Sitafloxacin was concluded that the method was found to be simple, reliable, selective, reproducible and accurate. The method was successfully used for quality control analysis of Sitafloxacin in bulk and Pharmaceutical dosage forms.

Keywords: Sitafloxacin (SFX), RP-HPLC, Validation.

INTRODUCTION

Sitafloxacin is an orally active of fourth generation new fluoroquinolone antibiotic. The chemical name of the Sitafloxacin is 7-[(4S)-4-Amino 6-azaspiro [2, 4] heptan-6 yl]-8-chloro-6-fluoro-1-[(2S)-2-fluorocyclopropyl]-4-oxoquinoline 3-carboxylic acid [1, 2]which includes amino pyrrolidine substituent at C-7 and a fluorocyclopropyl group at N-1. It contains a chlorine substituent at position C-8 of the quinolone nucleus and its molecular formula is C19H18ClF2N3O3. It is very high activity against gram-positive, gram-negative, and anaerobic organisms [3]. It has more potentactivity against gram-positive and gram-negative bacteria compared to currently available quinolones such as Ofloxacin, ciprofloxacin,to sufloxacin, and sparfloxacin[4, 5, 6 ] and reduced toxicity compared to other fluoroquinolones. The structure of Sitafloxacin shown in Figure 1. SFX is active against methicillin-resistant staphylococci, Streptococcus pneumoniae and other type of streptococci with reduced susceptibility to levofloxacin and other fluoro quinolones. The doses are given once daily because the post-antibiotic effect lasts greater than 6 hours and the serum half-life is 7 hours [7, 8]. Some of the recently developed quinolones have shown excellent anti-chlamydial activity in vitro and in vivo, as well as inclinical studies [9]. Only one analytical method was reported in the literature, such as determination of sitafloxacin in human plasma by using LC-MS/MS [10]. However literature survey reveals that, there is no method for estimation of sitafloxacin in bulk and pharmaceutical dosage forms by RP-HPLC. So we have developed and validated this method used for routine quality control analysis of Sitafloxacin.

Fig. 1: Chemical structure of SFX

MATERIALS AND METHODS

Materials

Sitafloxacin pure drug was obtained as a gift sample from Daiichi Sankyo Co., Ltd, Japan. HPLC grade Acetonitrile, and water [filtered through 0.2µ filters] were purchased from Merck, Mumbai, India.

Preparation of Solutions

Stock solution

100 mg of Sitafloxacin pure drug was dissolved in 100 ml of HPLC grade water: Acetonitrile (70:30) to get a concentration of 1000 μg/mL.

Standard solution

10 ml of stock solution was taken in 100 ml volumetric flask and diluted up to the mark with HPLC grade water to get a concentration of 100μg/mL.

Sample solution

20 tablets of Sitafloxacin were powdered and an amount of the powder equivalent to 100 mg of the drug was accurately weighed and transferred to the 100 ml volumetric flask, made up to the volume with diluent. The solution was placed in an ultrasonicator for 20 minutes and filtered through a 25 mm, 0.45 μm nylon syringe filter. 10 ml of this solution was taken and diluted to 100 ml by using a HPLC grade water to get a final concentration of 100 μg/mL. Five replicate sample solutions were prepared in the similar manner.

Hplc instrumentation and conditions

0.0.1 Instrumentation

Waters HPLC system consisting of WATERS 2695 separation module, an inbuilt auto sampler, column oven and WATERS 2996 (PDA) detector was employed for throughout the analysis. Chromatography was performed on a Symmetry C18 column. A sonerex sonicator was used for sonication. The data were acquired by using the EM Power2 software and other details of the instrumentation are given in Table 1.

Optimized chromatographic conditions

Chromatography was performed on a Symmetry C18 column using mobile phase containing mixture of Water: Acetonitrile 70:30% V/V. The mobile phase was filtered through membrane filter (0.45 µm), and vacuum degassed by sonication prior to use.

Table 1: Instrumentation and Optimized chromatographic conditions for proposed method

S. No. 0.0.1 Instrumentation Optimized Chromatographic Conditions
1 HPLC Waters: 2695 Separation Module
2 Detector Waters: 2996 PDA
3 Column Symmetry C18 (5 µm, 100X4.6 mm)
4 Column temperature 300C
5 Flow rate 0.9 mL/min
6 Injection volume 20 µL
7 Wavelength 300 nm
8 Run time 10 minutes
9 Diluent Water (HPLC Grade)
10 Mobile phase composition Water: ACN 70:30% V/V

The pump pressure and run time was maintained at 1500-2000 psi and 10 minutes respectively. Chromatography was performed at ambient temperature with flow rate at 0.9 mL/min and detection was carried out at 300 nm. Instrumentation and optimized chromatographic conditions for proposed method details are shown in Table 1.

RESULTS AND DISCUSSION

Validation study of Sitafloxacin

The Method validation was performed as per ICH guidelines for the determination of Sitafloxacin in bulk, and dosage forms. The method was validated with respect to parameters including accuracy, precision, linearity, robustness, specificity, system suitability, LOD and LOQ [11].

Assay of Sitafloxacin tablets

The developed method was applied to the assay of SFX tablets. The drug content was estimated with an average of six determinations, and its results were given in Table 2. The results were similar to the labeled claim of market formulations. The standard and sample chromatograms of Sitafloxacin were shown in Figure 2 and 3 receptively.

Table 2: Assay results of Sitafloxacin formulations

S. No. Formulations Standard

Peak area

 Sample

Peak area

 Labeled

Amount(mg)

 Amount

Found(mg)

 %Assay ±RSD*
1 M. F 1318938 1318929 100 99.96 99.96±0.12

* Average of 6 determinations


Fig. 2: RP-HPLC Chromatogram of Sitafloxacin


Fig. 3: RP-HPLC chromatogram of Sitafloxacin formulation (Tablets)

Specificity

The specificity of the proposed method was established by injecting the placebo and mobile phase solution in triplicate and the chromatograms were recorded. Comparison of chromatograms revealed that there were no interactions between the placebo and sample peaks. Finally, it was indicated that the method was specific.

Accuracy

The accuracy was determined by calculating the recovery of Sitafloxacin at 50%, 100%, and 150% and they were added to pre quantified sample solution. Recovery studies were carried out in the tablet in triplicate each in the presence of placebo. The mean percentage recovery of SFX at each level was not less than 99%, and not more than 102%. The percentage recovery of Sitafloxacin was found to be in the range of 100 to 101%. The results are shown in the Table 3.

Precision

Precision should be investigated by using authentic, and homogeneous samples. The Precision of this method was expressed as S. D and %RSD of series of repeated measurements. Precision of SFX determination by proposed method was ascertained by repeated analysis of homogeneous samples of Sitafloxacin standard solutions in the intraday under the similar conditions. The system precision and method precision results were shown in Table 4.

Linearity

Linearity of the proposed method was established by using series of standard solutions of Sitafloxacin at concentration levels from 5 to 25 μg/mL, like 5, 10, 15, 20, and 25 μg/mL. These linearity studies are repeated in triplicate with different stock solutions.

Graph 1: Linearity Graph of Sitafloxacin

The curve obtained by concentration on x-axis and peak area on y-axis against showed linearity in the concentration range of 5-25 μg/mL of SFX and linearity graph is shown in Graph 1. The regression equation was found to be Y = 98824x + 11140 with correlation coefficient is r2 = 0.9999. The Linearity and statistical analysis of data is shown in Table 5.

Robustness

The robustness was evaluated by the analysis of Sitafloxacin under different experimental conditions such as slight changes in chromatographic conditions like change of temperature (± 50C), flow rate (± 0.2 mL/min), and mobile phase composition (± 5%). It was distinguished that there were no changes in the chromatograms, and the parameters were within the limits, which indicates that the method was robust and suitable for routine use. The complete results are shown in Table 6 and the method is having good system suitability.

System suitability

This test was conducted on freshly prepared SFX standard solution and was used for the evaluation of the system suitability parameters such as retention time, area, USP tailing and theoretical plates, limit of detection and limit of quantification.

Five replicate injections for a system suitability test were injected into the chromatographic system and results were given in Table 7.

Table 3: Recovery data for the proposed RP-HPLC method

S. No. Concentration level Amount added (μg/mL) Amount found (μg/mL) Area obtained Mean

%Recovery ± S. D*

 %RSD*
1 50% 5 5.00 1908799 100±0.57 0.627
4.97 1956765
5.03 1999014
2 100% 10 9.98 2566768 100.13±0.67 0.664
9.97 2663052
10.09 2513953
3 150% 15 15.01 3349282 100.06±0.27 0.266
14.97 3278576
15.05 3331193

*S. D & %RSD is standard deviation and percentage of relative standard deviation

Table 4: System and Method Precision results of the proposed RP-HPLC method

 

S. No.

 

Injections

System Precision

Method Precision

Retention Time

Peak Area

Retention Time

Peak Area

1

1

2.207

1283904

2.190

1281340

2

2

2.198

1262428

2.198

1333464

3

3

2.207

1287461

2.198

1298485

4

4

2.207

1294271

2.207

1287461

5

5

2.198

1283464

2.207

1283904

6

6

2.207

1283921

2.207

1283854

7

MEAN

2.204

1282574.83

2.2011

1294751.33

8

SD

0.004

10685.87

0.007

19909.99

9

%RSD

0.210

0.833

0.319

1.537

Table 5: Linearity and Statistical analysis data for Sitafloxacin

S. No.

Concentration (µg/mL)

Area

Average Area

Statistical Analysis

Slope

Y-Intercept

Correlation Coefficient

1

5

520374

 

7478620

 

98824

 

11140

 

0.9999

2

10

992290

3

15

1500786

4

20

1989536

5

25

2475634

Table 6: Robustness results of the proposed RP-HPLC method

S. No.

Parameters

Peak Areas

Retention Time (Rt)

USP

Optimized

Used

Plate Count

Tailing Factor

 

1

 

Flow rate (±0.2)

 

0.9 mL/min

0.7

1495177

2.465

2639

1.396

0.9

1318938

2.198

2615

1.423

1.1

1166289

2.023

2491

1.442

 

2

 

Temperature (±50c)

 

300c

25

1468912

2.421

2594

1.394

30

1318938

2.198

2615

1.423

35

1167859

2.018

2569

1.685

 

3

Mobile phase composition (± 5%)

 

70:30

65:35

1412799

2.357

2608

1.393

70:30

1318938

2.198

2615

1.423

75:25

1179849

1.973

2457

1.648

Limit of Detection: The limit of detection (LOD) has established the minimum concentration at which the analyte can be reliably detected. LOD is determined by the signal to noise ratio and generally acceptable detection limit ratio is 3:1 and it was found to be 0.429 μg/mL.

Limit of Quantification: The limit of quantification (LOQ) has established the minimum concentration at which the analyte can be reliably quantified. LOQ is determined by the signal to noise ratio and a typical signal to noise ratio is 10:1 is acceptable for estimating the quantification limit and it was found to be 1.415 μg/mL. Finally the proposed method is having good system suitability and its parameters were shown in Table 7.

Table 7: System suitability parameters of proposed RP-HPLC method

S. No. Parameters Values
1 Wavelength (λ max) 300 nm
2 Linearity range(μg/mL) 5-25
3 Regression equation Y=98824x+11140
4 Correlation coefficient(r2) 0.9999
5 Retention time (minutes) 2.198
6 Theoretical plates 2615
7 Tailing factor 1.423
8 Limit of Detection (μg/mL) 0.429
9 Limit of Quantification (μg/mL) 1.415
10 Asymmetry Factor 1.672
11 Capacity factor (k) 0.147

CONCLUSION

A RP-HPLC method for quantitative estimation of SFX in bulk and pharmaceutical dosage forms is established. The method is simple, accurate, linear, sensitive and reproducible as well as economical for the effective quantitative analysis of SFX in bulk and tablet dosage forms. The method was validated, and all the method validation parameters were tested and shown to yield satisfactory results. The method is free from interactions of the other ingredients and excipients used in the tablet formulations. Finally it is concluded that the method is suitable for use in the routine quality control analysis of Sitafloxacin in API and in pharmaceutical dosage forms.

CONFLICT OF INTERESTS

Declared None

ACKNOWLEDGEMENTS

The authors would like to thank the management of Daiichi Sankyo Co., Ltd, Japan for the gift sample of drug used in this investigation.

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