1.INTRODUCTION:

Cefpodoxime proxetil (CEF) 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] (is shown in fig 1), is a third generation cephalosporin anti-biotic. It is used for infections of the respiratory tract, urinary tract and skin and soft tissues. It has greater activity against Staphylococcus aureus^[^2]. CEF is official in IP^[^3] and USP^[4], which describes liquid chromatography method for its estimation. Literature survey reveals the high-performance thin layer chromatography (HPTLC)^[^5] method for the determination of CEF individually. Literature survey also reveals the reversed-phase high-performance liquid chromatographic (RP-HPLC)^[^6] and spectrophotometric ^[7] methods for determination of CEF with other drugs. Dicloxacillin (DCX) is chemically 9 (2S,5R,6R)-6-[3-(2,6-dichlorophenyl)-5-methyl-1,2-oxazole-4-amido]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0] heptane-2-carboxylic acid (is shown in figure 2).^[8], and is a penicillinase resistant penicillin, used in the treatment of bacterial infections such as pneumonia and bone, ear, skin and urinary tract infection ^[9] .

It is official in IP ^[10] and USP ^[11], and describes RP-HPLC method for its estimation. Literature survey reveals HPLC^[^12] method for determination of DCX in pharmaceutical dosage forms as well as in biological fluids. Literature survey also reveals spectrofluorimetric^[^13] and RP-HPLC^[14]^-[16] methods for determination of DCX with other drugs.

The combined dosage form of CEF and DCX are available in the market for the treatment of infections caused by susceptible microorganisms like urinary tract infections and gonococcal urethritis. The combination of these two drugs is not official in any pharmacopoeia, hence, no official method is available for the simultaneous estimation of CEF and DCX in their combined dosage forms. Literature survey does not reveal any simple spectrophotometric or other method for simultaneous estimation of CEF and DCX in combined dosage form. The present communication describes simple, sensitive, rapid, and accurate RP-HPLC method for simultaneous estimation of both drugs in their combined tablet dosage forms.

Fig.No.1.Chemical structure of Cefpodoxime proxetil

Fig.No.2.chemical structure of Dicloxacillin

A literature survey reveals that there are few analytical methods reported for the estimation of Cefpodoxime Proxetil alone and in combination with Haloperidol ^{[11, 12]} or in combination with other antihypertensive drugs. However the reported methods have several limitations. In one of the reported method retention time for HPD was not found to be significant which limits its use and in another; flow rate for separation of both the drugs found to be >1ml/min which means excess of solvent is required throughout the analysis compared to usual flow rates (1±0.2ml/min) which is ideal for good column performance. Therefore in order to overcome the drawbacks of the reported methods; need arise to develop a new method which should be suitable for routine analysis of these drugs in combination. The present study is able to overcome the drawbacks in the sense of being economical and with significant retention time for both the drugs which proves that present method is perfect compared to reported methods.

2.EXPERIMENTAL:

2.1 MATERIALS AND METHODS

Reagents and chemicals

Methanol HPLC grade was procured from E. Merck Ltd, Mumbai. Methanol, orthophosphoric acid, Triethyl amine buffer AR grade were procured from S.D. fine chemicals, Hyderabad. Water HPLC grade was prepared using Millipore purification system. Cefpodoxime Proxetil and Dicloxacillin Sodium reference standards procured fro m Dr. Reddy’s Laboratories, Hyderabad.

Instrumentation

The HPLC system consists of water Empower 2695 having photodiode array detector system, which was connected with the help of Empower-2 software for data integration and processing. Xbridge ODS‐3V (250 X 4.6 mm) 5μ column was used for the analysis.

HPLC conditions

The contents of the mobile phase were consisting of methanol: water in the ratio of 50:50. These were filtered through 0.45μ membrane filter and degassed by sonication before use. The flow rate of mobile phase was optimized to 1.0 ml / min. The run time was set at 10 min and column temperature was maintained at ambient. The volume of injection was 10μl, and the eluent was detected at 290nm. Each of standard and test preparations was injected into the column and the responses recorded.

Preparation of stock solution:

The solution was prepared by dissolving 20.0 mg of accurately weighed Cefpodoxime Proxetil RS and 25.0 mg Dicloxacillin Sodium RS in Mobile phase, in two 100.0 mL volumetric flasks separately and sonicate for 20min. From the above solutions take 10.0 mL from each solution into a 50.0 mL volumetric flask and then makeup with mobile phase and sonicate for 10min.

Preparation of working standard solution:

The stock solutions equivalent to 20ppm to 80ppm with respect to both drugs were prepared in combination of Cefpodoxime Proxetil and Dicloxacillin Sodium as above, sonicated and filtered through 0.45µ membrane.

Preparation of sample drug solution for pharmaceutical formulations:

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 20 mg Dicloxacillin Sodium l and 25 mg Cefpodoxime Proxetil was weighed and dissolved in the 70 mL mobile phase with the aid of ultrasonication for 20 min. The content was diluted to 100 mL with mobile phase to furnish a stock test solution. The stock solution was filtered through a 0.45 µm Nylon syringe filter and 10.0 mL of the filtrate was diluted into a 50.0 mL volumetric flask to give a test solution containing 40 µg/mL Cefpodoxime Proxetil and 50 µg/mL Dicloxacillin Sodium

Procedure for calibration curve:

The contents of the mobile phase were filtered before use through 0.45micron membrane and pumped from the respective solvent reservoirs to the column at a specified flow rate.Prior to injection of the drug solutions, the column was equilibrated for at least 30min with the mobile phase flowing through the system. The chromatographic separation was achieved using a mobile phase consisting of Methanol : water at 50:50V/V the eluent was monitored using Pda detector at a wavelength of 290nm.The column was maintained at ambient temperature (27⁰c) and an injection volume of 20µl of each of standard and sample solutions were injected into the HPLC system to get the chromatograms. The retention time, peak areas of drug was recorded graph was plotted by taking concentration of the drug on x-axis and peak area on y-axis. A typical chromatogram of Dicloxacillin Sodium and Cefpodoxime Proxetil combination.

Table 1. Optimized chromatographic conditions.

Parameters	Chromatographic Conditions
Mobile phase	Methanol : Water ( 50 : 50 )%V/V
Stationary phase (column)	Inertsil -ODS C₁₈(250 x 4.6 mm, 5 µ)
Detection wavelength (nm)	290nm
Flow rate (ml/min)	1.0 ml/min
Volume of injection loop (µl)	20µl
Column temperature (°C)	Ambient
Run Time ( min )	7min
Drug RT (min)	2.9min for Dicloxacillin Sodium and 3.5 for Cefpodoxime Proxetil

Calculation:

The amount of drugs present in each pharmaceutical formulation was calculated by using the standard calibration curves (concentration in ppm was taken on x-axis and peak area on y-axis). A typical chromatogram of Dicloxacillin Sodium and Cefpodoxime Proxetil(100ppm) (formulation)

Optimized Chromatographic Conditions:

A Xbrdge C₁₈ [4.6 x 150 mm] column was used for the separation of drugs. The mobile phase comprised of water: Methanol(50_:50) .Injection volume was 20μl and run time was 15min and flow rate 1.0 ml/min. The column was maintained at ambient temperature and the eluent was detected at 290nm. The separation of CEF and DCX under optimized condition is shown in Figure 3.

Method:

Preparation of stock solution:

Reference solution: The solution was prepared by dissolving 20.0 mg of accurately weighed Cefpodoxime Proxetil RS and 25.0 mg Dicloxacillin Sodium RS in Mobile phase, in two 100.0 mL volumetric flasks separately and sonicate for 20min. From the above solutions take 10.0 mL from each solution into a 50.0 mL volumetric flask and then makeup with mobile phase and sonicate for 10min.

Preparation of working standard solution:

Preparation of sample drug solution for pharmaceutical formulations:

Procedure for calibration curve:

The contents of the mobile phase were filtered before use through 0.45micron membrane and pumped from the respective solvent reservoirs to the column at a specified flow rate. Prior to injection of the drug solutions, the column was equilibrated for at least 30min with the mobile phase flowing through the system. The chromatographic separation was achieved using a mobile phase consisting of Methanol: water at 50:50V/V the eluent was monitored using PDA detector at a wavelength of 290nm.The column was maintained at ambient temperature (27⁰c) and an injection volume of 20µl of each of standard and sample solutions were injected into the HPLC system to get the chromatograms. The retention time, peak areas of drug was recorded graph was plotted by taking concentration of the drug on x-axis and peak area on y-axis. A typical chromatogram for the linear responses of Dicloxacillin Sodium and Cefpodoxime Proxetil combination was shown in Fig 4 and 5.

Fig.No.04. calibration curve of Cefpodoxime Proxetil

Fig. No. 05. calibration curve of Dicloxacillin Sodium

2.6 System suitability

System suitability is a pharmacopoeial requirement and is used to verify, whether the resolution and reproducibility of the chromatographic system are adequate for analysis to be done. The tests were performed by collecting data from 5 replicate injections of standard solutions. The values obtained demonstrated the suitability of the system for the analysis of this drug combination and the system suitability parameters fall within ±2% standard deviation range during performance of the method. Here tailing factor for peaks of CEF and DCX was less than 2% and resolution was satisfactory.

3. RESULTS AND DISCUSSION:

The chromatographic conditions were optimized to develop RP-HPLC method for simultaneous determination of CEF and DCX with adequate resolution and rapid analysis time.

3.1 Method Validation^[^17]

The developed chromatographic method for simultaneous estimation of CEF and DCX was validated according ICH guidelines for linearity, accuracy, precision, specificity, robustness and ruggedness.

3.1.1 Linearity

According to USP; tablet powder equivalent to 20, 30, 40, 50, 60, 70 and 80 µg/ml of label claim was taken and dissolved in Methanol diluted appropriately with Methanol to obtain a concentration in the range of 20-80 µg/ml of the test concentration. Each of this concentration was injected to get reproducible response. The calibration curve was plotted as concentration of the respective drug versus the response at each level. The proposed method was evaluated by its correlation coefficient and intercept value calculated in the statistical study.

3.1.2 Recovery

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out using standard addition method at 50, 100 and 150 % level; known amount of standards was added to reanalyzed sample and subjected them to the proposed HPLC method. Percentage recovery was calculated from the amount found and actual amount added. The mean recovery is within acceptable limits which indicate that the method is accurate. The results of recovery studies are shown in Table 2

3.1.3 Precision

The precision of an analytical method is expressed in terms of SD or RSD of series of measurements. It was ascertained by replicate estimation of CEF and DCX by proposed method. Percentage relative standard deviation (%RSD) was found to be less than 2% which proves that method is precise. The results of precision study are shown in Table 3.

Preparation of standard stock solution

Standard stock solutions Cefpodoxime Proxetil and Dicloxacillin Sodium of strength 1mg/ml were prepared using dichloromethane. Appropriate amounts of these stock solutions were then further diluted to get the required concentrations of standard stock solutions.

Table 2:Recovery Studies.

Drug

Sample

No.

Amount present

(mg/ml)

Amount added

(mg/ml)

Amount estimated*

(mg/ml)

% Recovery*

S.D

R.S.D

CEF

2.06

3.0

6.0

9.0

5.0486

8.99

11.97

97.94

98.71

99.22

0.6433

0.6533

DCX

40.09

2.5

5.0

7.5

42.621

42.521

100.10

99.75

99.16

0.4750

0.4766

System suitability studies

The resolution, number of theoretical plates, retention time and peak asymmetry were calculated for the working standard solutions and is as shown in Table 2. The values obtained demonstrated the suitability of the system for the analysis of these drugs in combination.

Table 3: system suitability Studies.

Parameters	Cefpodoxime Proxetil	Dicloxacillin Sodium
Theoretical plates	10978.86	9550.677
Asymmetry Factor	1.05	1.15
HETP (cm)	0.00075	0.00162
Resolution*		4.63

ASSAY:

Preparation of sample solutions

Twenty tablets were weighed and powdered. Powder equivalent to 10 mg of Cefpodoxime Proxetil Sodium was weighed and transferred to 10 ml volumetric flask. Cefpodoxime Proxetil about 8 ml was added and sonicated for 10 min, volume was made up with the same solvent. This solution was then filtered through membrane filter paper. Further dilutions were made in dichloromethane to get concentrations in Beers law range. The retention times of Cefpodoxime Proxetil and Dicloxacillin Sodium were found to be 2.62 ± 0.02 and 3.96 ± 0.03 respectively. The assay was calculated from the equation of regression line for each drug. The percentage assay of individual drug was calculated.

CONCLUSION:

The results of the analysis of pharmaceutical dosage forms by the proposed methods are highly reproducible, reliable, and are in good agreement with the label claims of the drug. The additives usually present in the pharmaceutical formulations of the assayed samples did not interfere with Cefpodoxime Proxetil and Dicloxacillin Sodium. It may be said that the proposed methods are precise, sensitive, and accurate, so that these can be used as standard pharmacopoeial methods for the simultaneous determination of Cefpodoxime Proxetil and Dicloxacillin Sodium in tablets using the HPLC systems. The advantages of the proposed method involve a simple procedure for sample preparation and relatively short time of analysis. Apart from this, it can be used for assays of Cefpodoxime Proxetil and Dicloxacillin Sodium in biological fluids or in pharmacokinetic investigations.

ACKNOWLEDGEMENTS:

The authors would like to thank Ms Active Pharma Labs Pvt., Hyderabad, for providing the gift samples of Cefpodoxime Proxetil and Dicloxacillin Sodium for the project work. The authors are thankful to principal and Chairman of Teegala Ram Reddy College of Pharmacy, Hyderabad for their kind help and providing all necessary facilities.

REFERENCES:

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11. The United State Pharmacopeia. USP28-NF23. Rockville, MD: United States Pharmacopeial Convention, Inc.; 2005. p. 451-2.

12. Alderete O, González-Esquivel DF, Del Rivero LM, Castro Torres N. Liquid chromatographic assay for dicloxacillin in plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2004; 805:353-6.

13. Morelli B. Second-derivative spectrophotometric assay of mixtures of dicloxacillin sodium and ampicillin sodium in pharmaceuticals. J Pharm Sci 1988; 77:1042-6.

14. Barot T, Patidar K, Kshartri N, Vyas N. Development and validation of LC method for the determination of ampicillin and dicloxacillin in pharmaceutical formulation using an experimental design. Eur J Chem 2009; 6:955-64.

15. Kathiresan K, Murugan R, Hameed SM, Inimai GK, Kanyadhara T. Analytical method development and validation of cefixime and dicloxacillin tablets by RP-HPLC. Rasayana J Chem 2009;2:588-92.

16. Zhang MJ, Zhang HJ, Guan X, Xiao ZH. HPLC determination of dicloxacillin and amoxicillin in human plasma. Yaowu Fenxi Zazhi 2006; 26:228-31.

17. The International Conference on Harmonization, Q2 (R1) Validation of Analytical Procedure: Text and Methodology, 2005. Available from: http://www.ich.org/fileadmin/ Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf [Last Accessed on 2012 Dec 28].

Received on 12.11.2014 Accepted on 21.11.2014

Asian J. Res. Pharm. Sci. 4(4): Oct.-Dec. 2014; Page 155-159

Parameters

Chromatographic Conditions