A New Validated RP-HPLC Method for the Simultaneous Estimation of Cefpodoxime Proxetil and Ofloxacin in Bulk and Pharmaceutical Dosage Forms

 

Syeda Kulsum, Sravani A.*, M. Kanaka Durga, T. Snehalatha, Thimmareddy D.

Department of Pharmaceutical Analysis and Quality Assurance, Vijaya College of Pharmacy,

Munaganoor, Hayathnagar, Hyderabad

Corresponding Author E-mail: alamsravani@gmail.com

 

ABSTRACT:

A simple, rapid, specific, accurate and precise reverse phase high performance liquid chromatographic method was developed for the simultaneous estimation of Cefpodoxime proxetil and Ofloxacin Bulk and Tablet dosage form. An Thermo hypersil C18, 5 μ column having 150 x 4.6mm id in Isocratic mode with mobile phase containing 0.1M Dipotassium Hydrogen Phosphate Buffer : methanol  (90:10 %v/v pH: 6.0 adjusted with Ortho phosphoric acid) was used. The flow rate was 1ml/min and effluents were monitored at 265nm. The retention time of Cefpodoxime proxetil and Ofloxacin was 6.9min and 9.0min respectively. The concentration curves were linear in the concentration range of 200-600µg/mL and 200-600 µg/mL. The developed method was validated for specificity, precision, linearity, accuracy, LOD, LOQ, robustness. Recovery of Cefpodoxime proxetil and Ofloxacin in formulations was found to be in the range of 98% -102% and 98%-102% respectively confirms the non-interferences of the excipients in the formulation. Due to its simplicity, rapidness and good precision, the proposed HPLC method may be used for the simultaneous determination of these two drugs in pharmaceutical dosage forms. 

 

KEYWORDS: RP-HPLC, Cefpodoxime proxetil, Ofloxacin, Accuracy, Precision.

 


INTRODUCTION:

CEFPODOXIME PROXETIL(CEFO)  [1-3] (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyimino-acetyl]amino}-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid . Cefpodoxime is an oral third generation cephalosporin antibiotic. Cefpodoxime proxetil is a prodrug which is absorbed and de-esterified by the intestinal mucosa to Cefpodoxime. It is bactericidal and acts by inhibiting cell wall synthesis. It is active against gram positive and gram negative bacteria. It is commonly used to treat acute otitis media, pharyngitis, and sinusitis.

 

OFLOXACIN[4-7]  (RS)-7-fluoro-2-methyl-6-(4-methylpiperazin-1-yl)-10-oxo-4-oxa-1-azatricyclo[7.3.1.0]trideca-5(13),6,8,11-tetraene-11-carboxylic acid.  Ofloxacin is a synthetic chemotherapeutic antibiotic of the second generation flouroquinolone drug class It is bactericidal and acts on DNA gyrase and Topoisomerase. It is a broad spectrum antibiotic. It is active against gram positive organisms, gram negative and atypical bacteria. Apart from RTI they are also used for more resistant strains of bacteria and are especially useful in genitourinary infections like complicated urinary tract infections, invasive external otitis, bacterial prostates, cervicitis

 

Reason for the combination of Ofloxacin with Cefpodoxime proxetil [7,8]

Respiratory tract infection refers to any of a number of infectious diseases involving the respiratory tract.  Various antibiotics used in RTI like aminopencillins, cephalosporins, macrolides, fluoroquinolones. But when used individually these drugs have some limitations hence combination of Cefpodoxime proxetil and Ofloxacin is preferred because these have active against gram positive, gram negative and atypical bacteria and these have double attack on microorganisms since mode of action is different hence ensuring better cure.

 

Available formulation: Cepodem-O (Cefpodoxime proxetil200 mg, Ofloxacin200mg)

 

Several analytical procedures have been proposed for the quantitative estimation of Cefpodoxime proxetil and Ofloxacin separately and in combination with other drugs. HPLC and UV methods for estimation of Cefpodoxime proxetil alone [13, 14] and in combination with other drugs like Clavulanic acid [15, 16], Ambroxol Hydrochloride [17] and Dicloxacillin [18] are available. Similarly Ofloxacin alone[19] and in combination with other drugs like Ornidazole[20,21], Nitrozanide[22-24], Cefixime[25]  is estimated by UV and HPLC  have also been reported.

 

To our knowledge simple and rapid method for simultaneous determination of Cefpodoxime proxetil and Ofloxacin has not been reported so far. So attempt was taken to develop and validate a rapid reversed-phase high performance liquid chromatographic method for the quality control of Cefpodoxime proxetil and Ofloxacin in pharmaceutical preparations with lower solvent consumption along with the short analytical run time that leads to an environmentally friendly chromatographic procedure and will allow the analysis of a large number of samples in a short period of time. The method was validated and found to be accurate, precise and reproducible.

 

MATERIALS AND METHOD:

Apparatus

Waters e2695Alliance HPLC system connected with PDA Detector 2998 and Empower2 Software. The drug analysis data were acquired and processed using Empower2 software running under Windows XP on a Pentium PC.

Other Apparatus: Electronic balance, Sonicator, 0.45µ membrane filter

Reagents and chemicals

Pharmaceutical grade Cefpodoxime proxetil and Ofloxacin were kindly supplied as a gift sample by Dr. Reddy’s Laboratory, Hyderabad, Andhra Pradesh, India. Methanol was of HPLC grade and collected from E. Merck, Darmstadt, Germany. Dipotassium hydrogen phosphate was analytical reagent grade supplied by Fischer Scientific Chemicals. Water HPLC grade was obtained from a Milli-QRO water purification system.

 

Commercial formulation

Cefpodoxime proxetil and Ofloxacin Tablets available in the market as Cepodem-O in composition of Cefpodoxime proxetil (200mg), Ofloxacin (200mg). The samples were properly checked for their manufacturing license numbers, batch numbers, production, expiry dates and stored properly.

 

Preparation and selection of mobile phase

The preliminary isocratic studies on a reverse phase C18 column with different mobile phase combination of  Dipotassium hydrogen phosphate buffer pH6.0 and Methanol were studied for simultaneous separation of both the drugs. The optimal composition of mobile phase determined to be Buffer: Methanol (90:10 v/v) and filtered through 0.45µ membrane filter.

 

Preparation of standard solution

200mg Cefpodoxime proxetil and 200mg Ofloxacin was dissolved in 100 ml of diluent acetonitrile and was further diluted to get stock solution of Cefpodoxime proxetil and Ofloxacin (To get 400µg/ml and 400 µg/ml respectively). This is taken as a 100% concentration. Solution containing mixture of Cefpodoxime proxetil and Ofloxacin of five different concentrations (50%, 75%, 100% 125%, and 150% of target concentration) were prepared in the same way.

 

Preparation of sample solution

Sample solution containing both the drugs was prepared by dissolving tablet powder into diluent (acetonitrile). Ten tablets were weighed separately. Their average weights were determined. Powder of tablets equivalent to one tablet weight was weighed and taken in a 100 ml volumetric flask, dissolved in diluents, shaken and sonicated for about 20 minutes then filtered through 0.45µ membrane filter. The filtered solution was further diluted with the diluent to make the final concentration of working sample equivalent to 100% of target concentration.

 

Chromatographic conditions

The mobile phase, a mixture of Dipotassium hydrogen phosphate buffer and methanol (90:10v/v) pumped at a flow rate of 1 ml/min through the column (Thermo hypersil C18; 5μ, 150 X 4.6  mm) at 50şC.The mobile phase was degassed prior to use under vacuum by filtration through a 0.45μ membrane filter. Both drugs showed good absorbance at 265 nm, which was selected as wavelength for further analysis.

Validation of HPLC method

Method Validation

Method validation [9-12] is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. The newly developed RP-HPLC method was validated as per International Conference on Harmonization (ICH) guidelines for parameters like system suitability, linearity and range, precision (repeatability), intermediate precision (ruggedness), specificity, accuracy and robustness.

 

System suitability

System suitability study of the method was carried out by six replicate analysis of solution containing 100% target concentration of Cefpodoxime proxetil and Ofloxacin. Various chromatographic parameters such as retention time, peak area tailing factor, theoretical plates (Tangent) of the column and resolution between the peaks were determined and the method was evaluated by analyzing these parameters.

 

Specificity

The specificity of the method was determined by checking the interference of any components with that of analyte peak. Blank (only diluent) is injected to determine any interference.

 

Linearity

Linearity of the method was determined by constructing calibration curves. Standard solutions of Cefpodoxime proxetil and Ofloxacin of different concentrations level (50%, 75%, 100%, 125%, and 150%) were used for this purpose. Each measurement was carried out in six replicates and the peak areas of the chromatograms were plotted against the concentrations to obtain the calibration curves and correlation coefficients.

 

Accuracy (Recovery studies)

To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 50%, 100% and 150%. Known amounts of standard Cefpodoxime proxetil and Ofloxacin were added to pre-analyzed samples and were subjected to the proposed HPLC method.

 

Precision

Precision was evaluated by carrying out six independent sample preparation of a single lot of formulation. The sample solution was prepared in the same manner as described in sample preparation. Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise.

 

Robustness of method

To evaluate the robustness of the developed RP-HPLC method, flow and temperature variations were done. The effect of change in the flow and temperature were studied. The method was found to be unaffected by flow and temperature variation.

 

RESULTS AND DISCUSSION:

Optimization of the Chromatographic conditions

Optimization of mobile phase was carried out based on resolution, tailing factor and theoretical plates obtained for Cefpodoxime proxetil and Ofloxacin. During the trial runs both the drugs were tested with different mobile phase compositions like methanol: water, buffer: acetonitrile, buffer: methanol at various compositions and flow rates. The mobile phase consisting of buffer: methanol (90:10, v/v) at a flow rate of 1.0 ml/min was selected which gave sharp, well-resolved peaks for Cefpodoxime proxetil and Ofloxacin. The retention times for Cefpodoxime proxetil and Ofloxacin were 6.9 and 9.0 minutes, respectively. Both the drugs absorbed UV radiations appreciably at 265 nm, so the same was selected as the detection wavelength. The separation was carried out at room temperature.

 

Fig.1, 2, 3 represents the chromatograms of standard drugs, drugs in combined tablet formulation and blank mobile phase run, respectively

 


 

Figure 1:  Typical Chromatogram of standard Cefpodoxime proxetil and Ofloxacin


 

Name

Retention Time

Area

USP Resolution

s/n

USP Tailing

USP Plate Count

1

Cefpodoxime proxetil

6.9

3234261

 

771.8

0.87

3097

2

Ofloxacin

9.0

4453463

2.0

813.12

1.86

3074

 

 

Figure 2: Typical chromatogram of Cefpodoxime proxetil and Ofloxacin in marketed formulation.

 

Name

Retention Time

Area

USP Resolution

s/n

USP Tailing

USP Plate Count

1

Cefpodoxime proxetil

6.9

3232803

 

771.8

0.87

3138

2

Ofloxacin

9.0

4380646

2.0

813.12

1.85

3091

 

 

Figure 3 Typical chromatogram of Mobile phase blank run

 


Suitability System

Results of system suitability study are summarized in Table 1. Six consecutive injections of the standard solution showed uniform retention time, theoretical plate count, tailing factor and resolution for both the drugs which indicate a good system for analysis.

 

Table 1: Result of system suitability tests of Cefpodoxime proxetil and Ofloxacin

Parameters

Cefpodoxime proxetil

Ofloxacin

Linearity range

200-600µg/mL

200-600µg/mL

Correlation coefficient

0.999

1.0

Slope

30101x

41267x

Retention time

6.9

9.0

Resolution Factor

 

2.0

USP plate count

3138

3091

Tailing factor*

0.87

1.85

Limit of Detection(LOD)

10µg/mL

10µg/mL

Limit of quantification (LOQ)

30µg/mL

30µg/mL

*=%Mean

 

Chromatograms shown in figure1 and figure 2 explain that retention time for standard sample and commercial product of Cefpodoxime proxetil and Ofloxacin are same. This proves that, excipients have no effect on the analytical method. On the other hand, blank peak did not overlap drug peak. So the method is highly selective. A linear relationship between peak areas (average peak areas of six replicates) versus concentrations was observed for Cefpodoxime proxetil and Ofloxacin in the range of 50% to 150% of nominal concentration. Correlation coefficient was 0.999 for Cefpodoxime proxetil and 1.0 for Ofloxacin which proves that the method is linear. Calibration curve of Cefpodoxime proxetil and Ofloxacin are shown in Fig 4 and 5

 

Linearity:

 

Figure 4 Linearity of Cefpodoxime proxetil

 

Figure 5 Linearity of Ofloxacin

 

Results of accuracy study are presented in table 2. The measured value was obtained by recovery test. Spiked amount of both the drug were compared against the recovery amount.

 

% Recovery was 99% for Cefpodoxime proxetil and 99% Ofloxacin. All the results indicate that the method is highly accurate.

 

 


 

Accuracy

Table 2: Accuracy (%recovery) results of Cefpodoxime proxetil and Ofloxacin

No

Spiked Amount (mg)

Recovered Amount (mg)

%Recovered

%Average recovery

Sample

Cefpodoxime proxetil

99

1

20

19.8

99

2

40

39.6

99

3

60

59.4

99

Sample

Ofloxacin

 

1

20

19.8

99

99

2

40

39.6

99

3

60

59.4

99

 

 


Precision

Results of Intraday and inter day variability were summarized in table 3. Intraday variability was done from 9.00 am to 6.00 pm on the same day. % RSD of peak areas was calculated for various run .The method is precise as % RSD of peak area was less than 2% in all tests.

 

Table3: Intra day and inter day precision result of Cefpodoxime proxetil and Ofloxacin

Drug

%RSD

(intra-day)

%RSD (inter-day)

Cefpodoxime proxetil

1.05

1.15

Ofloxacin

1.07

1.32

Robustness

The results of robustness of the present method  have shown  that changes were made in the  flow rate and temperature did not produce significant changes in analytical results which are presented in Table 4 . As the changes are not significant we can say that the method is robust

 

Table 4: Results for robustness test of  Cefpodoxime proxetil and Ofloxacin

Parameter

Changes

Rt

USP Tailing

USP Plate Count

Cefpodoxime proxetil

Flow Rate

0.8

8.6

0.9

3393

1.2

5.8

0.8

3164

Analyst

Analyst1

6.9

0.8

3152

Analyst2

6.9

0.8

3177

Ofloxacin

Flow Rate

0.8

11.3

2

3063

1.2

7.6

1.7

3124

Analyst

Analyst1

9.0

1.8

3108

Analyst2

9.0

1.8

3076

CONCLUSION:

A validated RP-HPLC method has been developed for determination of Cefpodoxime proxetil and Ofloxacin in their bulk and combined tablet dosage forms. The method was found to be simple, accurate, precise and sensitive. The method was successfully applied for the determination of both drugs in combined tablet dosage form. In the future, this method may be applied for routine analysis of both the drugs in API, formulations, dissolution studies, bioavailability and pharmacokinetic studies.

 

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Reddy’s Laboratories, Hyderabad, Andhra Pradesh, India for providing the gift samples and to the Rainbow Pharma Training Lab, Kukatpally, Hyderabad, Andhra Pradesh, India for providing the research facilities.

 

Abbreviations/Acronyms

API: Active Pharmaceutical Ingredient

HPLC: High Performance Liquid Chromatography

RP- HPLC: Reverse Phase High Performance Liquid Chromatography

LOD: Limit of Detection

LOQ: Limit of Quantitation

Min: Minutes

RTI: Respiratory tract infection

RSD: Relative Standard Deviation

 

REFERENCES:

1.        http://www.drugbank.ca/drugs/DB0141

2.        http://www.medlineindia.com/antibiotic/cefpodoxime_proxetil.htm

3.        http://en.wikipedia.org/wiki/Cefpodoxime

4.        Indian Pharmacopoeia 2007, Govt. of India, The Indian pharmacopoeia commission, Ghaziabad, Ministry  of      Health and Family welfare

5.        http://www.drugbank.ca/drugs/DB01165

6.        http://en.wikipedia.org/wiki/Ofloxacin     

7.        Goodman and Gilman’s the pharmacological basis of therapeutics-11th edition (2006) by kaball.

8.        Martindale The complete drug reference.

9.        Sharma SK: Validation of pharmaceutical products and process. The Eastern Pharmacist 2001: 21-23.

10.     Linda LN: Reviewer guidance-Validation of chromatographic methods, Center for drug evaluation and research. 1994: 1-30.

11.     CH-Q2B, Validation of Analytical Procedures: Methodology. ICH Harmonized Tripartite Guideline, Geneva, 1996: 1-8.

12.     CH-Q2A, Text on Validation of Analytical Procedures. ICH Harmonized Tripartite Guideline, Geneva,   1995: 2-3. 

13.     Vasu Kumar Kakumanu et al. Development and validation of isomer specific RP-HPLC method for quantification of Cefpodoxime Proxetil. Journal of Chromatography B, Volume 835, Issues 1–2, 1 May 2006, Pages 16–20

14.     Ming-Juan Wang, et al, Comparison of Three RP-HPLC Methods for Analysis of Cefpodoxime Proxetil and Related Substances. Chromatographia, Volume 65, Numbers 1-2 (2007), 69-75

15.     S. Malathi et al, Simultaneous RP-HPLC Estimation of Cefpodoxime Proxetil and Clavulanic Acid in Tablets . Indian J Pharm Sci. 2009 Jan-Feb; 71(1): 102–105.

16.     Malathi S et al, Simultaneous RP-HPLC Estimation of Cefpodoxime Proxetil and Clavulanic Acid in Tablets . Indian J Pharm Sci.2009 Jan;71(1):102-5

17.     Rakesh Kotkar P et al, Development and Validation of RP-HPLC Method for Simultaneous Estimation of Cefpodoxime Proxetil and Ambroxol Hydrochloride in Bulk and in Tablets. International Journal of Research in Pharmaceutical and Biomedical Sciences, Vol. 3 (1) Jan – Mar 2012 ,156-163.

18.     Shraddha Patel et al, Development and validation of first order derivative spectrophotometric method for simultaneous estimation of Cefpodoxime proxetil and Dicloxacillin sodium in combined tablet. IJPRBS, 2012: Volume1 (2): 167-178  

19.     S Zen et al, High-performance liquid chromatography separation and quantitation of  Ofloxacin  enantiomers in rat  microsomes. Journal of Chromatography B: Biomedical Sciences and Applications, Volume 728, Issue 1, 14 May 1999, Pages 151–155

20.     Manisha Puranik et al, Simultaneous Determination of Ofloxacin and Ornidazole in Solid Dosage Form by RP-HPLC and HPTLC Techniques. Indian J Pharm Sci. 2010 Jul-Aug; 72(4): 513–517.

21.     B. Dhandapani et al, Method development and validation for the simultaneous estimation of Ofloxacin and Ornidazole in tablet dosage form by RP-HPLC. International Journal on Pharmaceutical and Biomedical Research ,Vol. 1(1), 2010, 42-48

22.     D.C. Premanand et al, A Validated RP-HPLC Method for Simultaneous Estimation of Nitazoxanide and Ofloxacin in Pharmaceutical Formulation. Der Chemica Sinica, 2010, 1 (2):1-5

23.     R. Sharma et al, Simultaneous RPHPLC Determination of Nitazoxanide and Ofloxacin in Combined Tablet Dosage Form. Indian J Pharm Sci. 2008 Jul-Aug; 70(4): 491–494.

24.     R Siva Kumar et al, Simultaneous RP-HPLC Estimation of Nitazoxanide and Ofloxacin in Tablet Dosage Forms. Asian J. Research Chem. 2(1): Jan.-March, 2009,43-45.

25.     Rajendran S.S, Simultaneous Estimation of Cefixime and Ofloxacin in Bulk and Tablet Dosage Form. Asian J. Pharm. Ana. 2011; Vol. 1: Issue 2, Pg 36-38

 

 

 

Received on 13.08.2012        Modified on 29.08.2012

Accepted on 07.09.2012        © AJRC All right reserved

Asian J. Research Chem. 5(9): September, 2012; Page 1166-1171