INTRODUCTION:

Cefpodoxime proxetil, chemically [(RS)-1 (isopropoxy carbonyloxy) ethyl (+)–(6R, 7R)-7[2-(2-amino-4-thiazolyl)-2(Z) methoxy imino acetamido]-3-methoxymethyl-8-oxo-5-thia-1-azabicyclo [4.2.0.]oct-2-ene-2-carboxylate], is a third generation cephalosporin antibiotic having activity against gram positive and gram negative micro organisms^[1]. Ofloxacin (OFL) is a fluoroquinolone derivative. Chemically, it is (±)-9-fluoro-2, 3-dihydro-3-methyl- 10-(4-methyl-1-piperazinyl) - 7-oxo-7H pyrido-[1, 2, 3-de]-1,4- benzoxazine-6 carboxylic acid. It is mainly used as antibacterial for the treatment of urinary tract infection and sexually transmitted diseases. Ofloxacin is official in USP¹, BP², IP³ and EP⁴ whereas Cefpodoxime is official in IP³, USP¹. Literature survey revealed that a number of analytical methods have been reported for estimation of Cefpodoxime proxetil in combination with other drugs i.e. spectrophotometry, HPLC⁶, RP-HPLC⁵, HPTLC⁷. The analytical methods reported for estimation of Ofloxacin in combination with other drugs include spectrophotometry, HPLC, RP-HPLC^8-11.

But not a single analytical method was traced for the simultaneous estimation of these two drugs in combined dosage form. So in this study two simple, accurate, precise, rapid and cost effective spectroscopic analytical methods are proposed for the simultaneous estimation of these two drugs in combined dosage form. The proposed methods were validated as per the ICH guidelines¹².

MATERIAL AND METHODS:

Apparatus:

Instrument used was an UV-Visible double beam spectrophotometer, SHIMADZU (model UV-1800) with a pair of 1 cm matched quartz cells. All weighing was done on SHIMADZU analytical balance (model AU-220).

Reagents and chemicals:

Pure drug samples of CP and OFL were obtained from reputed pharmaceutical company. Methanol LR was used as solvent. Calibrated glass wares were used throughout the work.

Marketed formulation:

Combined tablet formulation (Macpod Tab.) was procured from local market. Each tablet contains 200 mg CP and 200 mg OFL.

Preparation of standard solution:

Accurately weighed CP (10 mg) and OFL (10 mg) were transferred to two separate 100 ml volumetric flask, dissolved and diluted to the mark with Methanol to obtain standard solutions having concentration (100 μg/ml).6 ml of this standard solutions were transfered and diluted to 10 ml with methanol to prepare working standard solutions containing 6 μg/ml each.

Selection of analytical wavelength:

Standard solutions of CP and OFL (6 μg/ml) were scanned in the range of 200 to 400 nm for the determination of wavelength having maximum absorbance. Cefpodoxime proxetil shows 234.90 nm and OFL shows 298 nm as the wavelength having maximum absorbance. From the overlain spectra, Iso-absorptive point was found at 271.60 nm. So for the simultaneous equations method 234.90 nm and 298 nm were selected as analytical wavelengths. For the Q-absorbance ratio method, 271.60 nm and 298 nm were selected as analytical wavelengths.

Preparation of sample solutions:

Twenty tablets were weighed and powdered. The powder equivalent to 100 mg of CP and 100mg of OFL was transferred to 50 ml volumetric flask. Methanol (25 ml) was added to it and sonicated for 20 min. The solution was filtered through whatman filter paper No. 41 and the volume was adjusted up to the mark with Methanol.

Methods:

Method I: Simultneous Equation Method:

In simultaneous equation method, when no region can be found free from overlapping spectra of two chromophores, it is still possible to device a method based on measurements at two wavelengths. Two dissimilar chromophores must necessary have different powers of light absorption at some point or in linear absorption spectra.

If samples contain two absorbing drugs (X and Y), each of which absorbs at the λ_max of the other, it may be possible to determine both drugs by the technique of simultaneous equations.

From overlain spectra (Fig 1.) 234.90 nm λ_max for CP and 298 nm λ_max for OFL were selected for formation of simultaneous equation of two drugs. The absorbance at 234.90 nm and 298 nm for CP and OFL were measured. The absorptivity values of each drug at both wave-lengths were determined. The absorbance and absorptivity at this wavelength were substituted in following equations to obtain the concentration of both drugs.

…………… (1)

………...…… (2)

Where, A₁ and A₂ were absorbance of sample at 234.90 nm and 298 nm respectively

a_x1 and a_x2 are absorptivity of CP at 234.90 nm and 298 nm

a_y1 and a_y2 are absorptivity of OFL at 234.90 nm and 298 nm.

Figure.1: Overlain Spectra of Cefpodoxime (6µg/ml) and Ofloxacin (6µg/ml).

Validity of above framed equation was checked by using mixed standard of pure drug sample of two drugs, measuring their absorbance at respective wavelength and calculating concentration of two components.

Method II: Absorbance Ratio Method:

The absorbance ratio method is a modification of the simultaneous equation procedure. It depends on the property that, for a substance which obeys Beer’s Law at all wavelengths is a constant value independent of concentration or Path length. In the quantitative assay of two components in admixture by the absorbance by the absorbance ratio method, absorbance’s are measured at two wavelengths one being the λ_max of one of the components (λ₂) and the other being a wavelength of equal absorptivity of the two components (λ₁), that is , an isoabsorptive point.

From overlain spectra (Fig 1.) 271.60 nm (Isoabsorptive point) and 298 nm λ_max for OFL were selected for formation of Absorbance ratio equation of two drugs. The absorbance at 271.60 nm and 298 nm for CP and OFL were measured. The absorptivity values of each drug at both wavelengths were determined. The absorbance and absorptivity at this wavelength were substituted in following equations to obtain the concentration of both drugs.

C_X = ……….. (3)

C_Y = ……….. (4)

Q_m, Q_x, and Q_y were obtained as below:

Q_m = A₂/A₁,

Q_x = a_x2/a_x1,

Q_y = a_y2/a_y1

Where, A₁ and A₂ were absorbance of sample at 271.60 nm and 298 nm respectively,

a_x1 and a_x2 are absorptivity of CP at 271.60 nm and 298 nm,

a_y1 and a_y2are absorptivity of OFL at 271.60 nm and 298 nm.

Validation of the proposed method:

Linearity (Calibration curve):

The calibration curves were plotted over a concentration range of 2-10 mg/ml for each CP and OFL. Accurately measured standard stock solutions of each CP and OFL (0.2, 0.4, 0.6, 0.8 and 1.0 ml) were transferred to a series of 10 ml volumetric flask separately and diluted up to the mark with methanol. The absorbance of solution was then measured at 234.90 nm and 298 nm for simultaneous equation method and for absorbance ratio at 271.60nm and 298nm . The calibration curves were constructed by plotting absorbance versus concentration and the regression equations were calculated.

Method precision (repeatability):

The precision of the instrument was checked by repeated scanning and measurement of the absorbance of solutions (n = 3) of CP and OFL (6 mg/ml for both drugs) without changing the parameters of the proposed method.

Intermediate precision (reproducibility):

The intraday and interday precisions of the proposed method was determined by estimating the corresponding responses 3 times on the same day and on 3 different days over a period of one week for 3 different concentrations of standard solutions of CP and OFL (4, 6 and 8 mg/ml). The results were reported in terms of relative standard deviation (% CV).

Accuracy (recovery study):

The accuracy of the method was determined by calculating the recoveries of CP and OFL by the standard addition method. Known amount of standard solutions of CP and OFL were at added to prequantified sample solutions of CP and OFL (2 mg/ml for both drug). The amounts of CP and OFL were estimated by applying obtained values to the respective regression line equations.

Limit of detection and Limit of quantification:

The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines.

LOD = 3.3 × s/S

LOQ = 10 × s/S

Where, s = the standard deviation of Y-intercept of 3 calibration curves and

S =the mean slope of the 3 calibration curves.

Assay of tablet formulation:

Twenty tablets were weighed and crushed to obtain a fine powder. An accurately weighed sample equivalent to 100 mg of CP and 100 mg of OFL was taken in a stoppered volumetric flask (100.0ml); 40ml of methanol was added and sonicated for 10 min. The solution was filtered through whatman filter paper (No. 41) and the volume was made up to the mark with the same solvent. The aliquot portions of above solutions were further diluted with solvent to get final concentration of about 6 µg/ml CP and 6 µg/ml of OFL and absorbance were measured at 234.90 nm, 271.60 nm and 298.0 nm against blank. The concentrations of two drugs in sample were determined by using equations 1 and 2 and equation 3 and 4. The results are reported in the Table 2.

RESULS AND DISSCUTION:

The proposed method were validated as per ICH guideline. Methods discussed in the present work provide a convenient and accurate way for simultaneous analysis of CP and OFL. In simultaneous equation method, wavelengths selected for analysis were 234.9 nm (λ_max of cefpodoxime) and 298.0 nm (λ_max of ofloxacin). In Q analysis method, wavelengths selected were 271.60 nm (isoabsorptive point) (Fig. 4) and 298.0 nm (λ_max of ofloxacin). The plot of absorbance versus respective concentrations of CP and OFL were found to be linear in the concentration range of 2-10 µg/ml for both drug with correlation coefficient 0.999 at 234.90 nm and 0.999 at 298 nm as shown in Table 3 and Figure 2, 3. Precision was calculated in terms of repeatability, intraday and interday variations and %CV (coefficient of variance) was found to be in acceptance range (Table 3). The accuracy of method was determined by standard addition method. The % recovery ranges from 99.5-102 for CP and 98.04-98.33 for OFL in method 1 and 98.33-100.5 for CP and 98.6-100.8 for OFL in method 2. (Table 1).

Table 1: Recovery studies

Method	Spiked level (µg/ml)		Percent recovery % ±SD (n=3)
Method	CP	OFL	CP	OFL
I	4	4	102.10±0.20	98.30±0.10
II	4	4	98.33±0.15	100.8±0.15
I	6	6	101.10±0.083	98.33±0.23
II	6	6	100.13±0.10	99.5±0.15
I	8	8	99.5±0.219	98.04±0.99
II	8	8	100.50±0.09	98.60±0.35

Table 2: Results of simultaneous estimation of CP and OFL in marketed formulation

METHOD	Labelled mg/tablet		Obtained % of label claim^*
METHOD	CP	OFL	CP	OFL
I	200	200	99.00	99.53
II	200	200	101.1	98.26

Table 3: Validation parameters

	Simultaneous equation method				Q-absorbance ratio method
	CP		OFL		OFL	CP	(Iso)
	234.90 nm	298.00 Nm	234.90 nm	298.00 nm	298.00 nm	298.00 nm	271.60 nm
Linearity Range(µg/ml)	2-10	2-10	2-10	2-10	2-10	2-10	2-10
Slope	0.0319	0.0137	0.042	0.108	0.108	0.0137	0.022
Intercept	0.0142	0.0069	0.017	0.019	0.019	0.006	0.009
Correlation coefficient	0.9996	0.9987	0.9999	0.999	0.999	0.9996	0.999
LOD ((µg/ml)	0.319	0.225	0.00	0.867	0.390	0.376	0.439
LOQ(µg/ml)	0.967	0.682	0.00	0.629	0.367	0.682	1.332
Precision(%CV)
Repeatability	1.220	0.876	0.753	1.040	0.843	1.506	1.273
Interday(n=3)%	0.47	1.87	1.59	0.38	1.28	0.58	0.62
Intraday(n=3)%	0.41	1.38	1.48	1.13	0.80	0.16	1.34

Where, LOD = limit of detection,

Iso = Isoabsorptive point, LOQ = limit of quantification,

Figure.2: Calibration curve of standard CP at 234.90 nm

Figure.3: Calibration curve of standard OFL at 298 nm

This both methods can be successfully used for simultaneous estimation of CP and OFL in their combined tablet dosage form. Marketed tablets were analysed and results obtained were within the range of 98-102 % (Table 2).

CONCLUSION:

The low value of relative standard deviation for repeated measurement indicates that the method is precise. The value of % recovery is approximately 100%, which indicates that the methods can be used for estimation of these two drugs in combined dosage forms without any interference due to the other components present in the formulations. Hence proposed study presents two simple, accurate, precise, rapid and cost effective spectroscopic analytical methods are proposed for the simultaneous estimation of these two drugs in combined dosage form

REFERENCES:

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Received on 08.10.2011 Modified on 07.11.2011

Asian J. Research Chem. 4(12): Dec., 2011; Page 1836-1839