INTRODUCTION:

Cefpodoxime proxetil (Cef) is chemically (RS)-1(isopropoxycarbonyloxy) ethyl (+) - (6R, 7R) - 7 - [2 - (2 - amino - 4 - thiazolyl) - 2 - {(Z)methoxyimino}acetamido] - 3 – methoxy methyl - 8 - oxo - 5 - thia - 1 - azabicyclo oct-2-ene- 2-carboxylate and is official in United State Pharmacopoeia and British Pharmacopoeia^1,2. Potassium clavulanate (Pot.clav.) is monopotassium (Z)-(2R,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo heptane -2-carboxylate and is official in British Pharmacopoeia². Cef is active against a wide-spectrum of Gram-positive and Gram-negative bacteria. It is stable in the presence of beta-lactamase enzymes. As a result, many organisms resistant to penicillins and cephalosporins, due to their production of beta-lactamase, may be susceptible to Cef. The bactericidal activity of Cef results from its inhibition of cell wall synthesis. It is used in the treatment of acute otitis media, typhoid fever, pharyngitis and tonsillitis .The similarity in chemical structures allows the Pot. clav. to act as a competitive inhibitor of beta-lactamases secreted by certain bacteria which helps to restore the antimicrobial activity of Cef ^3-5.

Literature survey reveals several methods such as, U.V. spectroscopy^6-9, HPLC^10-18, HPTLC^19,20whichhave been reported for the estimation of individual drugs as well as in combination with other drugs. Not a single UV, HPLC or HPTLC method is reported so far for the simultaneous analysis of Cef and Pot.clav. in their combined dosage form. Here an attempt has been made to develop simple, rapid and accurate spectroscopic methods for simultaneous estimation of Cef and Pot.clav. in its formulation.

Materials and Methods:

A Shimadzu UV/Visible spectrophotometer, model 1700 (Japan) was employed with spectral bandwidth of 2 nm and wavelength accuracy of ± 0.5 nm, with automatic wavelength correction employing a pair of quartz cells. A Shimadzu electronic analytical balance (AX-200) was used for weighing the sample. An ultrasonic cleaner (Art No.400014CL) was used for sonicating the tablet sample solution.

Chemicals and reagents:

Analytical pure samples of Cef and Pot. clav. (Emcure Pharmaceutical, India) were used in the study. The pharmaceutical dosage form used in this study was Cepodem XP 325 tablet (Ranbaxy Pharmaceutical, India) labeled to contain 200 mg of Cef and 125 mg of Pot. clav. per tablet.

Standard stock solutions (for all three methods):

Standard stock solutions (100 µg/mL) of Cef and Pot.clav. were prepared by dissolving separately 10 mg of each drug each in 100 mL methanol: water mixture (60:40 v/v). The working standard solutions of these drugs were obtained by dilution of the respective stock solution with distilled water.

Sample stock solutions (for all three methods):

For the preparation of sample stock solutions, twenty tablets were weighed and crushed to fine powder. An accurately weighed powder sample equivalent to 10 mg of Cef was transferred to a 100 ml volumetric flask and dissolved in 50 ml of methanol: water mixture(60:40).After the immediate dissolution, the volume was made up to the mark with the same solvent. The solution was sonicated for about 30 min. and was then filtered through Whatmann filter paper No.41. The solution was suitably diluted with distilled water to obtain sample solutions containing Cef and Pot.clav. in the concentrations ratio of 10:6.25 µg/mL respectively.

Method A- Simultaneous equation method:²¹

Standard stock solutions (100 μg/mL) of Cef and Pot.clav. were prepared by dissolving separately 10 mg of drug each in 100 mL methanol: water mixture(60:40) .For forming the simultaneous equations, 233.0 nm (λ_maxof Cef) and 312.0 nm (λ_maxof Pot.clav.) were selected as the two working wavelengths. Fig.1 represents the overlain UV spectra of Cef and Pot.clav. Cef and Pot.clav.exhibited linearity with absorbances in the range of 1.5-50 ug/mL at their respective selected wavelengths. Co-efficients of correlation were found to be 0.998 and 0.999 for Cef and Pot.clav. respectively.The optical characteristics and regression values for the calibration curves are presented in table 1.

For simultaneous estimation of Cef and Pot.clav., mixed standards containing 10 ug/mL Cef and 6.25 ug/mL Pot.clav. were prepared by appropriate dilution of the standard stock solutions. The absorbances of the mixed standard solutions were measured at the selected wavelengths. A set of two simultaneous equations were established using the mean of absorptivity coefficients of Cef and Pot.clav. at the selected sampling wavelengths.

A1= 37.37 × C_CEF + 15.32 × C_{POT.CLAV. …….} (1) at 233.0 nm

A₂= 10.43 × C_CEF + 11.57 × C_{POT.CLAV. ….......}(2) at 312.0 nm

Where, 37.37 and 10.43 are the mean absorptivity values of Cef at 233.0 nm and 312.0 nm respectively.

15.32 and 11.57 are the mean absorptivity values of Pot.clav. at 233.0 nm and 312.0 nm respectively.

A₁ and A₂ are absorbances of mixed standards at 233.0 nm and at 312.0 nm respectively

C_CEF and C_POT.CLAV. are concentrations in g/ L.

The concentration of C_CEF and C_POT.CLAV in mixed standard and tablet formulation can be obtained by solving equation(1) and (2).

Fig. 1: Overlain Spectra of Cef and Pot.clav. in Simultaneous Equation Method and Multicomponent Mode Method.

Method B – Multicomponent mode of analysis:²¹

For the analysis of Cef and Pot.clav. by multicomponent method of analysis, the multicomponent mode of the UV visible spectrophotometer was used. Standard stock solution (100 ug / mL) of Cef and Pot.clav. were prepared by dissolving separately 10 mg of each drug in methanol: water mixture (60:40). For multicomponent method of analysis, 233.0 nm and 312.0 nm were selected as the two sampling wavelengths for Cef and Pot.clav.respectively.The drugs showed linearity in the concentration range of 1.5-50 ug/mL for both with regression coefficient (r²) values of 0.998 and 0.999 for Cef and Pot.clav. respectively.

Table- 1: Optical Characteristics and Validation Data of Cefpodoxime proxetil and Potassium clavulanate

Parameters

Cefpodoxime proxetil

Potassium clavulanate

Method-A

Method-B

Method-C

Method-A

Method-B

Method-C

Working wavelengths

Beer-Lamberts Law

range (μg/mL^-1)

Precision*

Interday (%RSD)

Intraday (%RSD)

LOD (μg/ml)*

LOQ (μg/ml)*

Regression Values:

I. Slope*

II. Intercept*

III. Regression coefficient (r²)*

233.0 nm

1.5-50

0.721

0.797

0.071

0.215

0.035

0.011

0.998

233.0 nm

1.5-50

0.545

0.283

0.071

0.215

235.0- 231.0 nm

1.5-50

0.563

0.539

0.018

0.053

0.142

0.061

0.998

312.0 nm

1.5-50

0.724

0.716

0.295

0.894

0.011

0.001

0.999

312.0 nm

1.5-50

0.720

0.532

0.295

0.894

314.0-310.0 nm

1.5-50

0.681

0.410

0.091

0.275

0.044

0.011

0.999

* Denotes average of six estimations; Where, Method-A – Simultaneous equation method; Method-B - Multi-component mode method; Method-C – Area under curve method

Six mixed standards in the ratio of 10:6.25 ug/mL of Cef and Pot.clav. respectively were prepared by appropriate dilution of standard stock solutions (100 ug/mL).

In multicomponent mode of the instrument, the mixed standards were scanned over the range of 190-400 nm at the selected sampling wavelengths. The overlain spectra (Fig.1) of the six mixed standards were then employed to determine the concentration of the drugs in sample solutions by analysis of the spectral data of sample solution with reference to that of mixed standards.

Method C – Area under curve method:²¹

Standard stock solutions (100 ug/mL) of Cef and Pot.clav. were prepared by dissolving separately 10 mg of each drug in 100 mL methanol: water mixture(60:40). For forming the simultaneous equations for Area under curve method (AUC), 235.0-231.0 nm (λ₁-λ₂) and 314.0-310.0 nm (λ₃-λ₄) were selected as the two sampling wavelength intervals. Fig 2 represents overlain UV spectra of Cef and Pot.clav.

Fig. 2: Overlain Spectra of Cef and Pot.clav. in Area Under Curve Method.

Cef and Pot.clav. exhibited linearity in the concentration range of 1.5-50 ug/mL at their respective selected wavelength intervals. Co-efficients of correlation were found to be 0.998 and 0.999 for Cef and Pot.clav. respectively. For simultaneous estimation, mixed standards containing Cef and Pot.clav. in the ratio of 10:6.25 ug/mL respectively were prepared by appropriate dilution of the standard stock solutions. The AUC of mixed standard solutions were measured at the selected wavelength intervals. A set of two simultaneous equations were established using the mean of absorptivity coefficients of Cef and Pot.clav. at the selected wavelength intervals.

A₁ = 153.9C_CEF + 48.66C_POT.CLAV. ---------------------- (3) at 235.0-231.0 nm (λ₁-λ₂)

A₂ = 44.58C_CEF + 42.65C_POT.CLAV. ---------------------- (4) at 314.0-310.0 nm (λ₃-λ₄)

Where – 153.9 and 44.58 are mean absorptivity values of Cef at λ₁-λ₂and λ₃-λ₄respectively.

48.66 and 42.65 are mean absorptivity values of Pot.clav. at λ₁-λ₂and λ₃-λ₄respectively.

A₁ and A₂ are the absorbance of mixed standard at λ1-λ₂and λ₃-λ₄respectively. C_CEF and C_POT.CLAV. are concentrations in g/L. The concentration of C_CEF and C_POT.CLAV in mixed standard and tablet formulation can be obtained by solving equation(3) and (4).

Assay of tablet formulation by method A, B and C:

Twenty tablets were accurately weighed and a quantity of tablet powder equivalent to 10 mg of Cef and 6.25 mg of Pot.clav. was weighed and dissolved in 100 ml of methanol: water mixture(60:40) with the aid of ultrasonication for 30 min. The solution was then filtered through Whatmann filter paper No.41 and diluted further to obtain final concentration of ratio of 10:6.25 ug/mL of Cef and Pot.clav. respectively. Solutions were scanned in the range of 400-200 nm .For the Simultaneous equation method (Method A), the absorbances of solutions were recorded at 233.0 nm (lmax of Cef) and 312.0 nm (lmax of Pot.clav.) and the concentrations of each drug in sample solutions were calculated using equations (1) and (2).For the multicomponent method, the concentrations of each drug were obtained by the spectral data of sample solutions with reference to that of the pure mixed standards. Areas under curve of sample solutions were recorded at the selected wavelength intervals ( 235.0-231.0 nm for Cef and 314.0-310.0 nm for Pot.clav) for the Area under the curve method.The concentrations of each drug in sample solutions were calculated using equations (3) and (4). The analysis procedure was repeated six times for all the methods with tablet formulation.

The results of the analysis and statistical validation data of the tablet formulation are given in table 2.

Table-2: Statistical Validation Data of Tablet Formulation

Methods	Tablet content	% Amount found*	% RSD *
A	Cef	100.76	0.871
A	Pot.clav.	100.85	0.923
B	Cef	100.80	0.771
B	Pot.clav.	100.21	0.516
C	Cef	100.75	0.614
C	Pot.clav.	100.34	0.743

* Denotes average of six estimations

Tablet Formulation: Cepodem XP 325, Ranbaxy pharmaceutical, India

Where, Method-A – Simultaneous equation method

Method-B - Multi-component mode method

Method-C – Area under curve method

Cef- Cefpodoxime proxetil

Pot.clav.- Potassium clavulanate

Validation of developed methods:

The accuracy of the proposed methods were determined by performing recovery studies at 80%, 100% and 120% of the test concentration as per ICH guidelines²². The basic concentration level of sample solution selected for spiking of the drugs standard solution was 10 μg/ mL of Cef and 6.25 μg/ mL of Pot.clav. The statistical validation data of recovery study is given in table 3.

Table-3: Statistical Validation of Recovery Studies

Level of % recovery	Methods	% Recovery*		%RSD*
Level of % recovery	Methods	Cef	Pot.clav.	Cef	Pot.clav.
80	A	99.99	100.05	0.147	0.132
	B	100.25	99.24	0.275	0.344
	C	99.94	99.97	0.055	0.187
100	A	99.88	100.13	0.077	0.333
	B	99.72	100.04	0.302	0.601
	C	99.90	100.03	0.100	0.092
120	A	100.43	100.14	0.459	0.437
	B	99.58	98.96	0.195	0.244
	C	100.02	99.76	0.058	0.041

* Denotes average of three estimations

Where, Method-A – Simultaneous equation method

Method-B- Multi-component mode method

Method-C – Area under curve method

Cef- Cefpodoxime proxetil

Pot.clav. - Potassium clavulanate

Under experimental conditions described, calibration curve, assay of tablets and recovery studies were performed. The developed methods were validated as per ICH guidelines for repeatability, intermediate precision (inter-day and intra-day precision studies), LOD, LOQ, slope, intercept, correlation coefficient as shown in table 1.

Also the results of the proposed methods were evaluated using Turkey-Kramer one way ANOVA to determine if there exist any significant difference between these methods for the analysis of Cef and Pot.clav., the results of which are shown in table 5.

Table -4: Ruggedness Study

Method	Parameter	% Mean		% R.S.D.*
Method	Parameter	Cef	Pot. clav.	Cef	Pot. clav.
Simultaneous Equation method	Instrument	99.71	98.76	0.784	0.250
Simultaneous Equation method	Analyst	99.48	99.35	0.424	0.541
Multico mponet Method	Instrument	99.87	99.63	0.382	1.017
Multico mponet Method	Analyst	99.08	99.12	0.700	0.491
Area Under Curve (AUC) Method	Instrument	99.77	100.15	0.555	0.788
Area Under Curve (AUC) Method	Analyst	99.65	99.31	0.254	0.435

*Denotes average of six estimations

Cef- Cefpodoxime proxetil

Pot.clav. - Potassium clavulanate

RESULTS:

The developed methods were validated as per ICH as shown in Table 1. The proposed methods were evaluated by the assay of commercially available tablets containing Cef and Pot.clav. (n=6) with mean % content of Cef and Pot.clav. were 100.77% and 100.47% with RSD values less than 2 respectively as shown in table 2. The mean % recoveries of Cef and Pot.clav. were 99.97% and 99.81% respectively (Table 3). The ruggedness of the developed methods was determined by evaluating the effect of change in instruments and analysts on the % mean content of drugs. The statistical validation data of ruggedness study given in table 4. The developed methods were compared statistically using Turkey-Kramer one way ANOVA which showed P>0.05. The calculated F value was 0.00664 and 1.198 for Cef and Pot.clav. respectively which is less than standard F value (table 5), indicating that there exists no significant difference between the three developed methods for the analysis of Cef and Pot.clav. in bulk and formulation.

Table-5: Turkey-Kramer method of ANOVA

For Cefpodoxime proxetil

One way Analysis of variance (ANOVA)

The P value is 0.9934 considered not significant.

F = 0.00664 = (MStreatment/MSresidual)

Variation among column means is not significantly greater than expected by chance.

Methods	Mean difference	P value
SE method Vs Multicomponent	-0.033	ns P>0.05
SE method Vs AUC method	0.017	ns P>0.05
Multicomponent Vs AUC method	-0.050	ns P>0.05

For Potassium clavulanate

One way Analysis of variance (ANOVA)

The P value is 0.3290 considered not significant.

F = 1.198 = (MStreatment/MSresidual)

Variation among column means is not significantly greater than expected by chance.

Methods	Mean difference	P value
SE method Vs Multicomponent	0.635	ns P>0.05
SE method Vs AUC method	0.505	ns P>0.05
Multicomponent Vs AUC method	0.130	ns P>0.05

DISCUSSION:

Cef and Pot.clav. are available in combined tablet dosage form used mainly for the treatment of acute otitis media. No single UV spectrophotometric method has been reported so far for the estimation of both the drugs in combination tablet dosage form. Here three simple UV spectrophotometric methods (Simultaneous equation method, multicomponent mode method and area under curve method) were developed for their simultaneous analysis. The RSD value for the tablet assay by these three methods is less than 2% as required by ICH guidelines. Also the % recovery was between 98-102% indicating high degree of accuracy of the proposed methods. The developed methods are simple, rapid, precise, accurate, rugged and can be employed for the routine estimation of Cef and Pot.clav. in both bulk and tablet dosage form.

ACKNOWLEDGEMENTS:

The authors express their gratitude to Pad. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pune, MH, India, for providing necessary facilities, and to Emcure Pharmaceutical, India, for the generous gift samples of pure Cef and Pot.clav.

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Received on 14.08.2011 Modified on 20.08.2011

Asian J. Research Chem. 4(10): Oct., 2011; Page 1600-1604