RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF CEFPODOXIME PROXETIL

RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF CEFPODOXIME PROXETIL

Deepshikha Patle ¹ and Ambadas Rangnath Rote ^{* 2}

Sardar Patel College of Technology ¹, Balaghat - 481001, Madhya Pradesh, India.

Pharmacy College Panchavati , Mumbai-Agra Road, Panchavati, Nashik - 422003, Maharashtra, India.

ABSTRACT: A new simple and rapid reverse-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for simultaneous estimation of cefpodoxime proxetil in bulk drugs and pharmaceutical dosage form. The separation was accomplished on a sunfire C-18 (250 × 4.6 mm, 5 μm) column with a mobile phase consisting of Methanol; Water (70:30 v/v) at a flow rate of 0.8 mL/min. The eluents were monitored at 236 nm by using PDA detector. The retention time for cefpodoxime proxetil was found to be 4.5 min, respectively. The cefpodoxime proxetil followed linearity in the concentration range of 5-30 μg/mL (r² = 0.998). The amount of drug estimated by proposed method was found to be in good agreement with label claim. The developed method was validated for sensitivity, accuracy, precision. The LOD and LOQ values were found to be 0.0004 and 0.0012 μg respectively for cefpodoxime proxetil. The proposed method can be used for routine analysis of both these drugs simultaneously in their combined dosage form.

INTRODUCTION: Cefpodoxime proxetil (CEFPO) Fig. 1 is chemically, 1-(isopropoxy carbonyloxy) ethyl (6R, 7R)-7-[2- (2-amino-4- thiazolyl)- (z) - 2- (methoxyimino) acetamido]-3-methoxymethyl-3-cephem-4-carboxylate ¹. It is a broad-spectrum third-generation cephalosporin antibiotic implicated in the treatment of upper respiratory tract, urinary tract infections ². The drug is official in IP ³ and USP ⁴. I.P and USP, describe liquid chromatography methods for its estimation. In literature, few RP–HPLC ^5-6, UV- Spectrophotometric ^7-9, and HPTLC ¹⁰ methods have been reported for determination of CEFPO in bulk drugs and pharmaceutical formulations.

Literature survey also reveals RPHPLC ^11-12 and spectrophotometric ¹³ methods for determination of CEFPO in combination with other drugs. No chromatographic method for estimation of Cefpodoxime proxetil in mobile phase (methanol: water at ratio (70:30) is reported. Hence an attempt has been made to develop a new RP-HPLC method using the following solvent at given mobile phase composition for simultaneous estimation in pharmaceutical dosage form.

MATERIAL AND METHOD:

Chemicals and Reagents: Analytical pure Cefpodoxime proxetil was obtained as gift sample from Vashu Enterprises Pvt. Ltd., Ludhiana, Panjab, India. The formulation Monocef-O-200 tablet (Aristo Pharmaceuticals Pvt. Ltd., Daman, India) was procured from the local market with labeled amount of 200 mg of Cefpodoxime proxetil Acetonitrile, methanol and water were used of HPLC grade, purchased from Fisher Scientific & S.D. Fine chem Ltd, India.

FIG. 1: MOLECULAR STRUCTURE OF CEFPODOXIME PROXETIL

Instrumentation and Chromatographic Condition: The HPLC system (YL-9100) consisted of following components: 2998 PDA detector, Rheodyne loop injector (7725 i) and 515 Binary pumps. Chromatographic analysis was carried out on a HiQsil C18 column (250mm × 4.6mm, 5µm, Kya Technologies corporation, JAPAN) Preparation of standard solution using mobile phase Methanol: Water (70:30) with flow rate of 0.8ml/min. Detection of eluent was made at 236 nm by PDA detector. The column was maintained at room temperature and injection volume of 20μl was used. The mobile phase was filtered through 0.45μm Chrom Tech Nylon-66 filter paper purchased from Pall India Pvt. Ltd, Daman, India.

Selection of Mobile Phase: The pure drugs of Cefpodoxime proxetil were injected into the HPLC system and run in different solvent systems. A mixture of different solvents like methanol: water, methanol: acetonitrile, acetonitrile: water were tried in order to determine optimum chromatographic conditions for effective separation of Cefpodoxime proxetil. After several permutation and combination, it was found that mixture of methanol: water gives satisfactory results as compared to other mobile phases. Finally, the optimal composition of the mobile phase methanol: water in the ratio of 70:30 (v/v) was selected, as it gave high resolution of Cefpodoxime proxetil with minimal tailing.

Preparation of Standard Solution: Standard stock solution of pure drugs was prepared by dissolving Quantity equivalent to 10 mg of the cefpodoxime proxetil in methanol to make 100 mL. This gave 100 µg/mL standard stock solution for cefpodoxime proxetil.¹⁴

Selection of Linearity Range and Preparation of Calibration Curve Standard: The above standard stock solution (100 mg/mL) of cefpodoxime proxetil was diluted with mobile phase to yield six calibration curve (CC) standards with concentrations of 5, 10, 15, 20, 25 and 30 µg/ mL.¹⁵

Analysis of Marketed Tablet Formulation: To determine the content of Cefpodoxime proxetil in marketed tablets (label claim 200mg per tablet). 20 tablets were weighed, and average weight was calculated. Tablets were triturated and powder equivalent to average weight was weighed. The drug was extracted from the tablet powder with 100 mL methanol. To ensure complete extraction it was sonicated for 15 min. 0.1 mL of supernatant was then diluted up to 10 mL with the mobile phase. The resulting solution was injected in HPLC and drug peak area was noted. A regression equation was generated using peak areas of standard solutions. Using the regression equation and peak area of the sample the amount of Cefpodoxime proxetil in the sample was calculated ¹⁶.

System Suitability: The system suitability was assessed by six replicate injections of the CEF containing 10 μg/ml of the drug. The resolution, peak asymmetry and the number of theoretical plates were calculated the obtained values were demonstrated the suitability of the system for the analysis of these drugs in combination ¹⁷.

Method Validation: The method was validated for linearity, accuracy, intraday and interday precision, LOD, and LOQ, in accordance with ICH guideline ¹⁸.

Linearity: obtained by conc. of 5,10,15,20,25 & 30 (µg/ml) of the standard solution of Cefpodoxime proxetil was transferred to series of 10 ml volumetric flasks and made up to volume with mobile phase. Each solution was injected and chromatogram was recorded. Retention time (mean ± s.d^a) of Cefpodoxime proxetil found to be 4.537 ± 0.003 respectively. The peak area of Cefpodoxime proxetil in each chromatogram was recorded s.d^a = standard deviation ^18-20.

TABLE 1: RESULTS OF ASSAY STUDY OF CEFPODOXIME PROXETIL

TABLE 2: SYSTEM SUITABILITY PARAMETERS FOR RP-HPLC

Accuracy: For the accuracy of proposed method recovery studies were performed. Recovery studies were carried out by standard addition of known amount of standard CFD to pre-analyzed tablet sample solution(10mg/ml) at three different levels 80%, 100% and 120% and the sample solution was analyzed by proposed method. For 80% recovery study amount of standard drug solution added was 8 mg/ml and in 100% and 150% recovery study the amount of standard drug solution added were 10 mg/ml and 12 mg/ml of CFD respectively. At each level of the amount, three determinations were performed ^{18, 21-22}. The results for recovery studies are given in Table 3.

Precision: Precision of proposed method was determined by repeatability and intermediate precision (Intraday and Interday) and was expressed in terms of S.D. and % R.S.D of the series of measurements. Repeatability of method was established by analyzing actual determination of six replicates of fixed concentration (10µg/ml) of CFD. Intraday precision was determined by analyzing three replicates of different concentration of standard CFD (10µg/ml, 15µg/ml and 20µg/ml) for three times in the same day. Interday precision was determined by analyzing replicates of three different concentration of standard CFD (10µg/ml, 15µg/ml and 20µg/ml) for the three consecutive days ^{18, 23}. For repeatability intra-day and interday precision, results are summarized in Table 4

TABLE 3: RESULTS OF ACCURACY STUDY

TABLE 4: SUMMARY OF VALIDATION PARAMETERS OF PROPOSED RP-HPLC

LOD= limit of detection; LO=limit of quantification; (%RSD) = % relative standard deviation

Limit of Detection (LOD) and Limit of Quantification (LOQ): They were calculated as 3.3 σ/S and 10 σ/S respectively. Where σ is the standard deviation of the response (y-intercept) and S is the mean of the slope of calibration plot ^{18, 24}.

RESULTS AND DISCUSSION:

Chromatographic (RP-HPLC) behavior of Cefpodoxime Proxetil: Chromatographic behavior of Cefpodoxime proxetil in different Composition of mobile phases.

Thus from the above, it has been observed that using a mobile phase of Methanol: Water (70:30 v/v) gave adequate retention at 4.533 min with good peak shape.

An RP-HPLC method was developed and validated for the determination of CEFO in tablet dosage forms on a column (Sunfire, c-18, 4.6 × 250mm, 5µm) with variable wavelength detection at 236 nm. The retention times of CEFO 4.53 min respectively. A linear correlation was obtained between area and concentrations of CEFO in the concentration ranges of 5-30 μg/ml. The low RSD values of CEFO for intraday (0.24-0.06 %) and interday (0.183-0.253%) at 236 nm. reveal that the proposed method is precise.

The limit of detection (LOD) and the limit of quantification (LOQ) for CEFO was found to be 0.00004 and 0.00012 μg/ml. These data show that the method is sensitive for the determination of Cefpodoxime proxetil. The recovery experiment was performed by the standard addition method. The mean recoveries were 98.4 ± 0.202 for CEFO. The results of recovery studies indicate that the proposed method is highly accurate. The proposed validated method was successfully applied to determine CEFO in tablet dosage form. No interference of the excipients with the absorbance of interest appeared; hence the proposed method is applicable for the routine simultaneous estimation of Cefpodoxime proxetil for pharmaceutical dosage forms.

FIG. 2: REPRESENTATIVE CHROMATOGRAM OBTAINED FOR STANDARD SOLUTION OF CEFPODOXIME PROXETIL (5 μg /ml, 4.542 ± 0.002 min)

FIG. 3: CALIBRATION CURVE FOR CEFPODOXIME PROXETIL                         

FIG. 4: CHROMATOGRAM OBTAINED FOR ASSAY STUDY OF CEFPODOXIME PROXETIL

CONCLUSION: The proposed method is highly sensitive, reproducible, specific, and rapid. The method was completely validated showing satisfactory data for all the method validation parameters tested. The developed method was robust in the separation and quantification of Cefpodoxime proxetil.

ACKOWLEDGEMENT: We would like to thank MGV’s Pharmacy College, Nashik-03 (university of Pune) for providing all necessary facilities during this research work.

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Patle D and Rote AR: RP-HPLC method development and validation of cefpodoxime proxetil. Int J Pharm Sci & Res 2014; 5(11): 4769-73. doi: 10.13040/IJPSR.0975-8232.5(11).4769-73.

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