Development and Validation of a Reverse Phase

Drug Invention Today ISSN: 0975-7619 Research Article www.ditonline.info

Development and Validation of a Reverse Phase-HPLC Method for the Determination of Balofloxacin in Bulk and Pharmaceutical Dosage Forms Naga Raju Potnuri*1, Devala Rao G 2 , Rajendra Prasad Y3 1Dept. of Pharmaceutical Analysis, Joginpally B.R. Pharmacy College, Yenkapally, Moinabad, R.R. Dist, AP, India 2K.V.S.R Siddhartha College of Pharmaceutical Sciences, Vijayawada, AP, India 3University Colleges of Pharmaceutical Sciences, Andhra University, Vishakhapatnam, AP, India A simple, precise, specific, sensitive and accurate reversed phase high performance liquid chromatography method was developed and validated as per the ICH guidelines for the quantitative determination of Balofloxacin in bulk and pharmaceutical formulations. The quantification was carried out by using Hypersil BDS C18-5µm, 150x4.6 mm column at ambient temperature with phosphate buffer (pH 2.5): Acetonitrile in the ratio of 70:30%v/v as a mobile phase, pH 2.5 adjusted with 0.1M ortho phosphoric acid at a flow rate is 1.0 ml/min. The eluent detection was carried out by using photodiode array detector at 293 nm. The retention time of Balofloxacin was 4.097min. The linearity was observed from 10150µg/ml with correlation coefficient is 0.999. The limit of detection and limit of quantitation were found to be 0.16µg/ml and 0.53µg/ml respectively. The Statistical analysis proves that the method was found to be simple, selective, reliable, accurate and reproducible. The method was successfully used for routine quality control analysis of Balofloxacin. Key words: Balofloxacin (BFX), RP-HPLC and validation.

INTRODUCTION

MATERIALS AND METHODS

Balofloxacin is an orally active of third generation fluoroquinolone antibiotic. The chemical name of the Balofloxacin1, 2 is 1-Cyclopropyl-6- fluoro-1, 4-dihydro-8 methoxy-7-[3-(methylamino)-1piperidinyl]-4-oxo-3quinoline carboxylic acid and its molecular formula is C20H24FN3O4. Balofloxacin is a fluoroquinolone that has a broader spectrum of activity and reduced toxicity than other fluoroquinolones and structure of Balofloxacin shown in Fig: 1. Balofloxacin is used for the treatment of urinary tract infection3. It exhibits excellent antibacterial activity against gram-positive bacteria such as multiple drug resistant staphylococci and pneumococci. BFX acts by binding and inhibiting TopoisomeraseII (DNA-gyrase) and Topoisomerase-IV enzymes, which are responsible for the coiling and uncoiling of DNA, which is needed for bacterial cell repair and replication4. Few analytical methods were reported in the literature, such as fluorescent spectroscopy5, RP-HPLC with fluorescence detection6, LC–EIMS7, luminescence spectroscopy8 methods have been developed for the determination of Balofloxacin either in bulk or tablet formulations. However, no reversed phase -HPLC method is available for estimation of Balofloxacin in bulk and pharmaceutical dosage form

Balofloxacin pure drug was obtained as a gift sample from Hetero Drugs Ltd., Hyderabad, Andhra Pradesh, India. BCin-100 mg tablets are manufactured by Hetero Labs Ltd., Himachal Pradesh, India and Marketed by Lupin Ltd., Mumbai, India. Bazucin-100mg tablets manufactured by Lupin Ltd., Mumbai, India were procured from local Pharmacy. HPLC grade Acetonitrile, methanol and water [filtered through 0.2µ filters] were purchased from Merck, Mumbai, India. Potassium dihydrogen phosphate and ortho phosphoric acid were purchased from Rankem, RFCL limited, New Delhi, India.

Fig. 1. Chemical structure of BFX

Preparation of Solutions Preparation of pH 2.5 Phosphate buffer solution: Dissolve 6.8g of potassium dihydrogen phosphate in 1000 ml of HPLC grade water (filtered through 0.2µ filters) and degassed. Adjust the pH to 2.5 by 0.1M ortho phosphoric acid. Preparation of stock solution: 100 mg of Balofloxacin pure drug was dissolved in 100ml of HPLC grade methanol: water (80:20) to get a concentration of 1000 µg/ml. Preparation of working 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. Preparation of sample solution: 20 tablets of Balofloxacin 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 methanol: water in the ratio of 80:20. The solution was placed in an ultrasonicator for 20min and filtered through a 25 mm, 0.45 µm nylon syringe filter. 10ml of this solution was taken

Corresponding Author: Naga Raju Potnuri, Joginpally B.R. Pharmacy College,Yenkapally (V), Moinabad (M), R.R. (Dist.), AP, India; e-mail: < [email protected] >

Received 15-10-2012; Accepted 16-11-2012 December, 2012

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Naga Raju Potnuri et al.: Development and Validation of a Reverse Phase-HPLC Method for the Determination of Balofloxacin in Bulk and Pharmaceutical Dosage Forms

HPLC Instrumentation And Conditions

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 similar manner.

Instrumentation: Waters HPLC system consisting of a WATERS 2695 separation module, an inbuilt auto sampler, a column oven and WATERS 2996 photodiode array detector was (PDA) employed for throughout the analysis. Chromatography was performed on a Hypersil BDS C185µm, 150x4.6 mm column. A digisum DI 707 digital pH meter used for pH adjustment and a bandline sonerex sonicator was used for sonication. The data were acquired using the EM Power-2 software and other details of the instrumentation are given in Table 1.

Table1: Instrumentation and Optimized chromatographic conditions for proposed method

S.NO

Instrumentation

1

HPLC

2 3 4 5 6 7 8

Detector Column Column temperature Flow rate Injection volume Wavelength Run time

9

Diluent

10

Mobile phase composition

Optimized chromatographic conditions Waters: 2695 Separation Module Waters: 2996 PDA Hypersil BDS-C18 300C 1.0 ml/min 20µL 293 nm 10 minutes Methanol :Water 80:20 (HPLC Grade) Phosphate Buffer (pH 2.5): ACN 70:30% v/v

Optimized chromatographic conditions: Chromatography was performed on a Hypersil BDS C18-5µm, 150x4.6 mm column using mobile phase containing mixture of Phosphate buffer (pH 2.5): Acetonitrile 70:30%v/v and adjusting its pH to 2.5 with 0.1M ortho phosphoric acid. The mobile phase was filtered through membrane filter (0.45 µm) and vacuum degassed by sonication prior to use. The pump pressure and run time was maintained at 1500-2000 psi and 10 min respectively. Chromatography was performed at ambient temperature (300C) under isocratic conditions at a flow rate is 1.0 ml/min and detection was done at 293 nm. Instrumentation and optimized chromatographic conditions for proposed method details are shown in Table 1.

Table 2: Assay results of Balofloxacin formulations S. No

1 2

Formulations

B-Cin (Lupin) Bazucin (Lupin)

Standard Peak area

Sample Peak area

Labeled Amount(mg)

Amount Found(mg)

%Assay ±RSD*

2831558 2831558

2769290 2801327

100 100

98.82 98.96

98.82±0.14 98.96±0.18

* Average of 6 determinations

Fig. 2. RP-HPLC Chromatogram of Balofloxacin;

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

Table 3: Recovery data for the proposed RP-HPLC method S. No

Concentration level

1

50%

2

100%

3

150%

Amount added (µg/ml) 5

Amount found (µg/ml)

5

4.98

1412487

5

5.08 9.96 9.90 10.18 15.21 14.96 15.21

1440669

10 10 10 15 15 15

5.00

Area obtained

Mean %Recovery ± S.D*

%RSD #

100.58±1.05

1.04

100.19±1.44

1.44

100.87±0.96

0.95

1418001

2821757 2804420 2882488 4307134 4235822 4306528

*S.D is standard deviation; # %RSD is percentage of relative standard deviation

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Naga Raju Potnuri et al.: Development and Validation of a Reverse Phase-HPLC Method for the Determination of Balofloxacin in Bulk and Pharmaceutical Dosage Forms

Table 4: System and Method Precision results of the proposed Reverse Phase-HPLC method System Precision S. No

1 2 3 4 5 6 7 8 9

Method Precision

Injections Retention Time (Rt) 4.249 4.028 4.097 3.986 4.230 4.313 4.151 0.132 3.184

1 2 3 4 5 6 MEAN SD %RSD

Peak Area

Retention Time (Rt)

2854567 2836544 2831558 2833209 2820107 2808617 2830767 15560.597 0.549

4.201 4.130 4.077 3.966 3.867 3.727 3.994 0.176 4.427

Peak Area 2876656 2805200 2795419 2866740 2864850 2867853 2846119.667 35849.144 1.259

Table 5: Linearity and Statistical analysis data for Balofloxacin S. No 1 2 3 4 5

Linearity level 10% 25% 50% 100% 150%

Concentration (PPM) 10 25 50 100 150

Area

Average area Slope

280179 683589 1367090 2806043 4066750

1533942

Statistical Analysis Y-Intercept Correlation coefficient (r2)

27319

8630

0.999

Table 6: Robustness results of Balofloxacin Parameters Optimized

S. No

1

Flow rate ( ±0.2)

USP

1 ml/min

2

Temperature ( ±50c)

300c

3

Mobile phase composition (± 5%)

70:30

Used

Peak areas 3673082 2831558

Retention time (Rt) 4.854 4.097

Plate count 3303 5149

Tailing factor 1.74 1.50

0.8 1 1.2 25 30 35 65:35 70:30 75:25

2385333 2926598 2831558 2972933 2810299 2831558 2721907

2.969 3.625 4.097 3.337 6.178 4.097 9.879

2707 2869 5149 2875 3279 5149 5911

1.73 1.78 1.50 1.73 1.04 1.50 1.40

RESULTS AND DISCUSSION Validation study of Balofloxacin: The Method validation was performed as per ICH guidelines for the determination of Balofloxacin in bulk and in the pharmaceutical dosage forms. The method was validated with respect to parameters including specificity, accuracy, precision, linearity, robustness, system suitability, limit of detection and limit of quantification.

Assay of Balofloxacin tablets: The developed method was applied to the assay of BFX tablets. The content was calculated as an average of six determinations and experimental results were given in Table 2. The results were very close to the labeled value of commercial tablets. The representative standard and sample chromatograms of Balofloxacin were shown in Figure 2 and 3 receptively. Specificity: The specificity of the Reverse phase-HPLC method was established by injecting the mobile phase and placebo solution in triplicate and recording the chromatograms. While the comparison of chromatograms there was no interference from the placebo with the sample peak. Therefore, it was concluded that the method is specific.

Graph 1: Linearity graph of Balofloxacin

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Accuracy (Recovery studies): The accuracy was determined by calculating the recovery of Balofloxacin at 10%, 25%, 50%, 100% and 150% was added to a pre-quantified sample solution. The recovery studies were carried out in the tablet in triplicate each in the presence of placebo. The mean percentage recovery of BFX at each level was not less than 99% and not more than 101%. The percentage recovery of Balofloxacin was found to be in the range of 100 to 101%. The results are shown in the Table 3. Precision: Precision should be investigated using homogeneous, authentic samples. Precision of analytical method was expressed as standard deviation and percentage

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Naga Raju Potnuri et al.: Development and Validation of a Reverse Phase-HPLC Method for the Determination of Balofloxacin in Bulk and Pharmaceutical Dosage Forms

of relative standard deviation of series of replicate measurements. Precision of BFX estimation by proposed method was ascertained by replicate analysis of homogeneous samples of Balofloxacin standard solutions in the intraday under the similar conditions. The system precision and method precision results were shown in Table 4. Table 7: System suitability results for proposed RP-HPLC method S.No 1 2 3 4 5 6 7 8

Injections 1 2 3 4 5 MEAN SD %RSD

Area 2836544 2831558 2833209 2820107 2808617 2826007 11520.99 0.407677

Retention Time 4.028 4.097 3.986 4.230 4.313 4.1308 0.137582 3.330629

Table 8: System Suitability Parameters of proposed RPHPLC method S. No 1 2 3 4 5 6 7 8 9 10

Parameters Wavelength (λ max) Linearity range(µg/ml) Regression equation Correlation coefficient(r2) Retention time (min) Theoretical plates Tailing factor Limit of detection (µg/ml) Limit of quantitation Capacity factor (k)

Values 293 nm 10-150 Y=27319x+8630 0.999 4.097 5149 1.50 0.16 0.53 1.0485

Linearity: Linearity of the proposed method was established by using series of standard solutions of Balofloxacin at concentration levels from 10 to 150%, like 10%, 25%, 50%, 100% and 150%. This linearity studies are repeated for three times with different stock solutions. The curve obtained by concentration on x-axis and peak area on y-axis against showed linearity in the concentration range of 10-150 µg/ml of BFX and linearity graph is shown in Graph 1. The regression equation was found to be Y = 27319x + 8630 with correlation coefficient is r2 = 0.999. The Linearity and statistical analysis of data is shown in Table 5. Robustness: The robustness was evaluated by the analysis of BFX under different experimental conditions such as slight changes in chromatographic conditions like change of flow rate (± 0.2 ml/min), temperature (± 50C), and mobile phase composition (± 5%). It was observed that there were no marked changes in the chromatograms and the parameters are within the limit, which indicates that the method has robustness and suitability for routine use. The complete results are shown in Table 6 and the method is having good system suitability. System suitability: The system suitability test was carried out on freshly prepared BFX standard solution (100%) was used for the evaluation of the system suitability parameters such as area, retention time, USP peak tailing, the number of theoretical plates, LOD and LOQ. Five replicate injections for a system suitability test were injected into the chromatographic system and system suitability of the system results are given in Table 7.

Limit of Detection (LOD): The limit of detection has established the minimum concentration at which the analyte can be reliably detected. LOD is determined by the signal to noise ratio and signal to noise ratio 3:1 is generally considered acceptable for estimating the detection limit and it was found to be 0.16µg/ml. Limit of Quantitation (LOQ): The limit of quantitation 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 quantitation limit and it was found to be 0.53µg/ml. Finally the proposed method is having good system suitability and system suitability parameters as shown in Table 8.

CONCLUSION A New RP-HPLC method for quantitative determination of BFX in bulk and in pharmaceutical dosage forms is established. This method is simple, reliable, linear, accurate, sensitive and reproducible as well as economical for the effective quantitative analysis of BFX in bulk and tablet formulations. The method was completely validated showing satisfactory data for all the method validation parameters tested and method is free from interference of the other active ingredients and additives used in the formulation. Therefore the method is suitable for use the routine quality control analysis of Balofloxacin in API or in pharmaceutical dosage forms.

ACKNOWLEDGEMENT The authors would like to thank the management of Hetero Drugs Ltd., Hyderabad, Andhra Pradesh, India for the donation of drug used in this investigation.

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Source of support: Nil, Conflict of interest: None Declared

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