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Available online at
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Research Article
A VALIDATED STABILITY INDICATING RP-HPLC METHOD
FOR SIMULTANEOUS ESTIMATION OF CEFPODOXIME AND
LEVOFLOXACIN IN BULK AND FORMULATION
B. Spandana, Ravi Pratap Pulla, K. Vanitha Prakash
Dept of Pharmaceutical Analysis and Quality Assurance, SSJ College of pharmacy, V.N.Pally,
Gandipet, Hyderabad, Telangana. 500075.
ABSTRACT
A simple, rapid, precise and accurate stability-indicating reverse phase HPLC method was developed and validated for simultaneous estimation
of Cefpodoxime proxetil and Levofloxacin in bulk and formulation. The method has shown adequate separation of Cefpodoxime proxetil and Levofloxacin from their degradation products. Separation was achieved on ACE (250 mm x 4.6 mm, 5 μ) column, kept at ambient temperature, using a mobile phase consisting of 0.01M Ortho phosphoric acid and Methanol (40:60) at a flow rate of 1.2 ml/min and UV detection at 275 nm. The average retention times for Cefpodoxime proxetil and Levofloxacin were found to be 3.068 and 3.870 min respectively. Cefpodoxime
proxetil, Levofloxacin and their combination drug product were subjected to acid hydrolysis, base hydrolysis, oxidation, thermal and photolytic
stress conditions. Validation of the method was carried out as per ICH guidelines. Linearity was established for Cefpodoxime proxetil and Levofloxacin in the range of 2.5-7.5 μg/mL . Correlation coefficient was found to be 0.9997 and 0.9994 for Cefpodoxime proxetil and Levofloxacin respectively. The percentage recovery of Cefpodoxime proxetil and Levofloxacin was found to be in the range of 98.0 - 102.0 %.
The method was found to be specific and stability indicating as no interfering peaks of degradants and excipients were observed.
Address for correspondence:
Dr. Ravi Pratap pulla
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INTRODUCTION
Simultaneous estimation of drug combination[1] is generally done by separation using chromatographic methods like HPLC, GC and HPTLC etc. These methods are accurate and precise with good reproducibility, but the cost of analysis is quite high owing to expensive instrumentation, reagent and expertise. Hence it is worthwhile to develop simpler and cost effective method for simultaneous estimation of drugs for routine analysis of formulation. Spectrophotometric analysis fulfills such requirement where the simultaneous estimation of the drug combination can be done with similar effectiveness as that of chromatographic methods. For the purpose of spectral analysis in order to relate chemical structure to electronic transitions, and for analytical situations in which mixture contribute interfering absorption, a method of manipulating the spectral data is called derivative spectroscopy[2]. Most of the drugs in multi component dosage forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. HPLC method eliminates tedious extraction and isolation procedures.
There is no stability indicating method reported for the simultaneous estimation of these drugs in a combined dosage formulation. Present work describes rapid, accurate, reproducible, and economical methods for simultaneous estimation of these drugs in tablet formulation.
Cefpodoxime is an oral third generation cephalosporin antibiotic. It is active against most Gram positive and Gram negative bacteria. It is commonly used to treat pharyngitis, and sinusitis. Cefpodoxime proxetil is a prodrug which is absorbed and de-esterified by the intestinal mucosa to Cefpodoxime
Cefpodoxime
Levofloxacin is a synthetic broad-spectrum antibacterial agent for oral and intravenous administration. Chemically, levofloxacin, achiral
fluorinated carboxyquinolone, is the pure (-)(S)-enantiomer of the racemic drug substance ofloxacin.
Molecular structure of Levofloxcin Hemihydrate
MATERIALS AND METHODOLOGY
Chemicals and reagents: Cefpodoxime and Levofloxacin standard drugs, Ortho phosphoric acid were obtained from lara drugs, kukatpally, hyderabad. Methanol and water used were HPLC grade (QUALIGENS). Commercially available tablets LEVOPOD were obtained from local market.
INSTRUMENT:
Waters HPLC 2e2695 series consisting pump, Auto sampler, photodiode array detector, Thermostat column compartment connected with Waters (alliance) Empower-2 software.
Chromatograhic Conditions:
The mobile phase consisting of 0.01% Ortho phosphoric acid and methanol (HPLC grade) in the ratio of 40:60v/v was pumped into the column at a flow rate of 1.2 mL/min. It was an isocratic elution. The column used was ACE, 4.6x250 mm, 5μ at 30°C. The detection was monitored at 275 nm using PDA detector and the run time was 7min.
Mobile Phase Preparation:
Mix 400 ml of Ortho phosphoric acid and 600ml of methanol in the ratio 40: 60 % v/v.
Standard Stock Solution Preparation:
Weigh and transfer 250mgof Levofloxacin & 200 mg of Cefpodoxime of working standard into 50 mL volumetric flask, add 10 mL of diluent and sonicated to dissolve and dilute to volume with diluent.
Standard Preparation:
Transfer5mL of standard stock solution into 25 mL volumetric flask and dilute to volume with diluent.
Sample Preparation:
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Observation:
In the above method, both Cefpodoxime and Levofloxacin are separated well with good resolution, good symmetrical factor. The theoretical plates observed for both the peaks are also within the range and the same are eluted within a run time of 10min. This method is suitable for Validation.
RESULTS AND DISCUSSION PRECISION
The relative standard deviation (%RSD) of the six assay preparations of CEFPODOXIME and LEVOFLOXACIN was calculated and it was found to be 0.11%.
S No
Name
Cefpodoxime proxitel Levofloxacin hemihydrates
RT Area RT Area
1 S-Precision-1 3.062 5724020 3.849 8222425
2 S-Precision-2 3.064 5720474 3.846 8226973
3 S-Precision-3 3.066 5727085 3.841 8227453
4 S-Precision-4 3.069 5728367 3.849 8227591
5 S-Precision-5 3.065 5728763 3.843 8221554
6 S-Precision-6 3.065 5728065 3.841 8223799
Average 3.065 5727805 3.845 8224580
Standard Deviation 0.00 17135.58 0.00 8797.58
% of RSD 0.04 0.59 0.04 0.34
Precision-1 Chromatogram of Recommended Procedure Using Sample Drug SAMPLE RETENTION
TIME
USP RESOLUTION USP TAILING PLATE COUNT
Cefpodoxime 3.065 --- 1.05 5608
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Drug Precision-1 Chromatogram of Recommended Procedure Using Sample drug ACCURACY
To study the accuracy of the method, recovery studies were carried out. To the formulation equivalent to 200 mg of Cefpodoxime and 250 mg of Levofloxacin at the levels of 50%, 100% and 150% was added to pure Cefpodoxime and Levofloxacin and made up to the mark with
Mobile phase and filtered through Whatmann filter paper and chromatograms were recorded. The concentration of drug present in resulting solution was determined using developed procedure and percentage recovery and percentage RSD were calculated.
Accuracy 50% -1 Chromatogram Using Sample Drug in 50 μg/mL
Accuracy 100% -1 Chromatogram Using Sample Drug in 100 μg/mL
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Standard Drug Chromatograms
Standard Chromatogram-1
Standard Chromatogram-2 ASSAY:
Assay values for precision
S.No Sample weight Sample-1
area
Sample-2 area
%Assay %Assay
1 999.5 5724020 8222425 99 100
2 999.5 5720474 8226973 99 100
3 999.5 5727085 8227453 99 100
4 999.5 5728367 8227591 99 100
5 999.5 5728763 8221544 99 100
LINEARITY
Aliquots of standard Cefpodoxime and Levofloxacin stock solution (0.2 ml to 0.8 ml ) (1ml=1000μg/mL) were taken in different 10 ml volumetric flasks and diluted up to the mark with the diluents such that the final concentrations of Cefpodoxime and Levofloxacin are in the range of 300-900 μg/mL.
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Linearity -50% ChromatogramLinearity -75% Chromatogram
Linearity -100% Chromatogram
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Linearity -150% ChromatogramLinearity values of Cefpodoxime and Levofloxacin
Conc in μg/mL
Standard Calibration curve of Cefpodoxime by RP-HPLC method 2861246
4299595
5726199
7153078
8580212
y = 16616x R² = 0.99
0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 8000000 9000000 10000000
0 20 40 60 80 100 120 140 160
Cefpodoxime Levofloxacin
S.No Sample name RT Area RT Area
1 LINEARITY-50% 3.064 2861246 3.843 4112188
2 LINEARITY-75% 3.060 4299595 3.841 6162389
3 LINEARITY-100% 3.062 5726199 3.834 8226608
4 LINEARITY-125% 3.058 7153078 3.827 10232407
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Co c i μg/ LStandard Calibration curve of Levofloxacin by RP-HPLC method
CEFPODOXIME LEVOFLOXACIN
CONC% Area ug/ml LOD LOQ CONC% Area ug/ml LOD LOQ
50 2861246 200 S/N 412 50 4112188 250 S/N 512
75 4299595 300.00 2.913 9.709 75 6162389 375 2.9297 9.7656
100 5726199 400.00 100 8226608 500
125 7153078 500 125 10232407 625
150 8580212 600 150 12319644 750.00
LIMIT OF DETECTION (LOD)
From the linearity data calculate the limit of detection and quantitation, using the following formula. LOD= 3.3 σσ = standard deviation of the response SS = slope of the calibration curve of the analyte. The limit of detection (LOD) & LOQ for
Cefpodoxime was found to be 2.913 & 9.70 .
LIMIT OF QUANTITATION (LOQ):
LOQ = 10 σ σ = standard deviation of the response SS = slope of the calibration curve of the analyte. The limit of detection (LOD) & LOQ for Levofloxacin was found to be 2.929 & 9.76.
4112188 6162389
8226608
10232407 12319644
y = 19288x R² = 0.99
0 2000000 4000000 6000000 8000000 10000000 12000000 14000000
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Chromatograms illustrating LOD of 0.5% Working StandardsChromatograms illustrating LOQ of 0.5% Working Standards
ROBUSTNESS
In order to prove that the method is robust, flow rate of the mobile phase (±0.2ml/min) and the column
temperature (±5ᵒc) are varied. The results showed that they have passed the system suitability parameters.
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Chromatogram of Cefpodoxime and Levofloxacin for robustness studies – Flow change (1.2ml/min)Chromatogram of Cefpodoxime and Levofloxacin for robustness studies – Temp-25ᵒC
Chromatogram of Cefpodoxime and Levofloxacin for robustness studies – Temp-35ᵒC
DEGRADATION PROFILE:
Acid: Tranfer 999.5 mg weight of sample into a 50 ml of volumetric flask and add 10 ml of 0.1n HCl and sonicate 30 min and add 10 ml of 0.1n NaOH make up with mobile phase. Transfer above solution 5 ml into 25 ml volumetric flask dilute to volume with mobile phase.
BASE: Transfer 999.5 mg weight of sample into a 50 ml volumetric flask and add 10 ml of 0.1N NaOH and sonicate 30 min and add 10 ml of HCl make up volume with mobile phase. Tranfer above solution 5ml into 25 ml volumetric flask dilute to volume with mobile phase.
PEROXIDE: Transfer 999.5 mg weight of sample into a 50 ml of volumetric flask and add 10ml peroxide and sonic make up volume with mobile
phase. Transfer above solution 5ml into 25 ml volumetric flask dilute to volume with mobile phase.
HEAT: Before sample weighing exposes the sample at 10535ᵒC. Transfer the 999.5 mg weight of sample into a 50 ml volumetric flask and add 15ml of mobile phase and sonicate 30 min and make up with mobile phase. Transfer above 5ml into 25ml volumetric flask dilute to volume with mobile phase.
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Chromatogram of Acid degraded Sample
Chromatogram of Base degraded Sample
Chromatogram of Heat degraded Sample
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Chromatogram of Peroxide degraded Sample
CONCLUSION
From the developed method it was found that both Cefpodoxime proxitel and Levofloxacin hemihydrate obey linearity within the concentration range of 2.5-7.5 g/mL. From the results of precision was found that the % RSD is less than 2, which indicates that the method has good reproducibility. From the results shown in accuracy it was found that the percentage recovery values of formulation from the placebo solution were in between 98.0 – 102.0 which indicates that the proposed method is accurate and also reveals that the commonly used excipients and additives in the pharmaceutical formulations were not interfering in the proposed method. From the results of Robustness it was found that a little variation in methods does not affect the intended use. From the results of Forced degradation studies it was found that method is fit to use stability studies in pharmaceutical routine analysis.
The proposed method was simple, sensitive and reliable with good precision and accuracy. The proposed method is specific while estimating the commercial formulations without interference of excipients and other additives. Hence, this method can be used for the routine determination of Cefpodoxime and Levofloxacin hemihydrate in pure samples and pharmaceutical formulations.
REFERENCES
1. http://www.drugbank.ca/drugs/DB01416
2.http://www.nlm.nih.gov/medlineplus/druginfo/med s/a698024.html
3. A Text Book of Pharmaceutical Analysis by David. G. Watson, 1999.
4. High pressure liquid chromatography, High priced liquid chromatography (pdf), Lecture5.HPLC. by Alexander Efimov.
5. International Conference on Harmonization, "Q2B: Validation of Analytical Procedures: Methodology; Availability," Federal Register 62(96), 27463–27467 (1997).
6. Bertram G. Katzung: Basic and Clinical Pharmacology, 8 th edition, 2001,
7. Wolff, M.E., ed. “Burger’s Medicinal Chemistry”, 5thedn., Wiley Interscience, New York, 1981, vol 5
8. www.Drugbank.com
9. ArgekarA. P. and SawantJ. G. Simultaneous Determination of Cefpodoxime Hydrochloride and Levofloxacin hemihydrate from Tablets by Ion Pair Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) , Informa healtProxitelare , 1999, Vol. 25, No. 8 , Pages 945-950
