Simultaneous UV Spectrophotometric Methods for Estimation of Cefixime Trihydrate and Ofloxacin in Bulk and Tablet Dosage Form

Audumbar Mali¹*, Santosh Jadhav¹, Rahul Gorad², Aamer Quazi², Ritesh Bathe¹, Manojkumar Patil¹, Ashpak Tamboli³

¹Department of Pharmaceutics, Sahyadri College of Pharmacy, Methwade, Sangola-413307, Solapur, Maharashtra, India.

²Department of Pharmaceutics, ASPM’S K. T. Patil College of Pharmacy, Osmanabad-413501, Maharashtra, India.

³Department of Pharmaceutical Chemistry, Sahyadri College of Pharmacy, Methwade, Sangola-413307, Solapur, Maharashtra, India.

*Corresponding Author E-mail: maliaudu442@gmail.com

ABSTRACT:

Simple, precise, economical, fast and reliable two UV methods have been developed for the simultaneous estimation of Cefixime Trihydrate and Ofloxacin in bulk and pharmaceutical dosage form. Method A is Absorbance maxima method, which is based on measurement of absorption at maximum wavelength of 287 nm and 296 nm for Cefixime Trihydrate and Ofloxacin respectively. Method B is area under curve (AUC), in the wavelength range of 265-301 nm for Cefixime Trihydrate and277-320nm for Ofloxacin. Linearity for detector response was observed in the concentration range of 5-25μg/ml for Cefixime Trihydrate and 5-25 μg/ml for Ofloxacin. The accuracy of the methods was assessed by recovery studies and was found to be 98.83% and 100.12% for Cefixime Trihydrate and 102.71 % and 99.01 % Ofloxacin by using method A and B respectively. The developed method was validated with respect to linearity, accuracy (recovery), precision and specificity. The results were validated statistically as per ICH Q2 R1guideline and were found to be satisfactory. The proposed methods were successfully applied for the determination of for Cefixime Trihydrate and Ofloxacin in commercial pharmaceutical dosage form.

KEY WORDS: Cefixime Trihydrate, Ofloxacin, Simultaneous estimation, Accuracy, Absorbance maxima method, Area under curve.

1. INTRODUCTION:

Cefixime Trihydrate (CEF) chemically is (6R, 7R)-7-[[(z)-2-(2-aminothiazol-4-yl) 2-[(carboxy methoxy) imino] acetyl]-amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2ene-2-carboxylic acid. It is an oral third generation of cephalosporin and is used as an antibacterial and especially against gram negative, gram positive and anaerobic bacteria pathogens including β- lactamase producing strains. It consists of high affinity for penicillin binding proteins with deceitful site of activity. It acts by inhibition of bacterial cell-wall synthesis. It is clinically used in the treatment of susceptible infections including gonorrhea, otitis media, pharyngitis, lower respiratory-tract infections such as bronchitis and urinary-tract infections.^1-4

Fig. 1: chemical structure of Cefixime Trihydrate

Ofloxacin (OFL) a fluorinated carboxyaquinolone, chemically is a racemate (+) -9-fluro-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 a synthetic broad spectrum antibacterial agent official in BP, USP and EP. Ofloxacin have much greater antibacterial activity towards urinary tract infections. It acts by inhibiting DNA gyrase of microorganisms.^5-8

Fig. 2: chemical structure of Ofloxacin

A survey of pertinent literature revealed thatin estimation of individual^9-11 as well as combination of Cefixime Trihydrate and Ofloxacin. Simultaneous determinations of Cefixime Trihydrateand Ofloxacindosage form were also reported like HPLC^12,13, RP-HPLC^14-16, HPTLC¹⁷ and UV-Spectroscopy^18-21. Therefore an attempt was made to develop a new rapid and sensitive UV Spectrophotometric method and to validate as per ICH-guidelines. A comprehensive literature research reveals the lack of a Spectrophotometric analytical method for simultaneous estimation of Cefixime Trihydrate and Ofloxacin in pharmaceutical formulations. A successful attempt was made to develop accurate, precise and simple method of analysis for estimation of both the drugs in combined dosage form.

2. MATERIALS AND METHODS:

2.1 Apparatus and instrumentation:-

A Shimadzu 1800 UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. Single Pan Electronic balance (Contech, CA 223, India) was used for weighing purpose. Sonication of the solutions was carried out using an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India).Calibrated volumetric glassware (Borosil®) was used for the validation study.

2.2 Materials:-

Reference standard of Cefixime Trihydrate and Ofloxacin API were supplied as gift sample by Cipla Pharmaceutical Company, Goa, India. The commercial formulation ABIXIM*O with label claim 200 mg Cefixime Trihydrate and 200 mg Ofloxacin per tablet were purchased from local market Mangalwedha, Dist:-Solapur, Maharashtra, India.

Fig. 3: It shows λmax of Cefixime Trihydrate

Fig. 4: It shows λmax of Ofloxacin

2.3 Method development: -

2.3.1 Preparation of standard stock solution: -

Stock solution was prepared by diluting 10 mg of each drug in sufficient quantity of methanol in separate volumetric flask and volume was made up to 100 ml to get the concentrations of 100 μg/ml for each drug. Dilutions from stock solution were prepared in the range of 5-25 μg/ml for Cefixime Trihydrate and 5-25 μg/ml for Ofloxacin. Methanol was used as a blank solution.

2.3.2 Method A: Absorption Maxima Method: -

For the selection of analytical wavelength, standard solution of Cefixime Trihydrate and Ofloxacin were scanned in the spectrum mode from 400 nm to 200 nm separately. From the spectra of drug λmax of Cefixime Trihydrate, 287 nm [Fig.3], and λmax of Ofloxacin,296 nm [Fig.4], were selected for the analysis. Aliquots of standard stock solution were made and calibration curve was plotted.^{22, 23}

2.3.3 Simultaneous estimation of Cefixime Trihydrate and Ofloxacin:

The wavelength maxima of Cefixime Trihydrate and Ofloxacin were determined and found to be 287 nm (λ1) and 296nm (λ2)respectively where there was no interference among the drugs. The overlain spectrum is shown in Fig.5.

Fig.5 Isobestic point of Cefixime Trihydrate and Ofloxacin

2.3.4 Method B: Area under Curve Method:

From the spectra of drug obtained after scanning of standard solution of Cefixime Trihydrate and Ofloxacin separately, area under the curve in the range of 265-301 nm and 277-320 nm was selected for the analysis. The calibration curve was prepared in the concentration range of 5-25 μg/ml for Cefixime Trihydrate and 5-25 μg/ml for Ofloxacin at their respective AUC range. Both drugs followed the Beer-Lambert’s law in the above mentioned concentration range. The calibration curves were plotted as absorbance against concentration of Cefixime Trihydrate and Ofloxacin. The coefficient of correlation (r), slope and intercept values of this method are given in Table 2.

Area calculation: (α+β) =

Where, α is area of portion bounded by curve data and a straight line connecting the start and end point, β i the area of portion bounded by a straight line connecting the start and end point on curve data and horizontal axis λ1 and λ2 are wavelength range start and end point of curve region.^24-26

2.3.5 Application of the proposed methods for the determination of Cefixime Trihydrate and Ofloxacin in tablet dosage form:

For the estimation of drugs in the tablet formulation, 20 tablets were weighed and weight equivalent to 200 mg of Cefixime Trihydrate and 200mg of Ofloxacin was transferred to 100 ml volumetric flask and ultrasonicated for 20 minutes and volume was made up to the mark with methanol. The solution was then filtered through a Whatman filter paper (No.42). The filtrate was appropriately diluted further.

In Method-A, the concentration of Cefixime Trihydrate and Ofloxacin was determined by measuring the absorbance of the sample at 287nm and 296nm respectively in zero order spectrum modes. By using the calibration curve, the concentration of the sample solution was determined.

In Method-B, the concentration of Cefixime Trihydrate and Ofloxacin was determined by measuring area under curve in the range of 265-301 nm and 277-320nm. By using the calibration curve, the concentration of the sample solution was determined.

Table 1: Table shows Results of Analysis of Tablet Formulation

Method	Drug	Label Claim mg	Sample Solution Concentration (µg/ml)	Amount found (%)*±	% Recovery	%RSD
A	Cefixime Trihydrate	400 mg	20	101.36±1.04	98.83	0.7125
B	Cefixime Trihydrate	400 mg	20	98.54 ±0.91	100.12
A	Ofloxacin	400 mg	20	100.59 ±1.74	102.71	0.72361
B	Ofloxacin	400 mg	20	98.71 ±1.60	99.01

*n=3, % RSD = % Relative Standard Deviation.

Fig.6: It shows AUC of Cefixime Trihydrate

Fig.7: It shows AUC of Ofloxacin

3. Validation of the developed methods: [27-31]

The methods were validated with respect to accuracy, linearity, precision and selectivity.

3.1 Accuracy:

Accuracy of an analysis was determined by systemic error involved. Accuracy may often be expressed as% Recovery by the assay of known, added amount of analyte. It is measure of the exactness of the analytical method. Recovery studies carried out for both the methods by spiking standard drug in the powdered formulations 80%, 100%, 120% amount of each dosage content as per ICH guidelines.

3.2 Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of Cefixime Trihydrate and Ofloxacin. Result should be expressed in terms of correlation co-efficient.

3.3 Precision:

The reproducibility of the proposed method was determined by performing tablet assay at different time intervals (morning, afternoon and evening) on same day (Intra-day assay precision) and on three different days (Inter-day precision). Result of intra-day and inter-day precision is expressed in % RSD.

3.4 Sensitivity:

The limit of detection (LOD) and limit of quantitation (LOQ) were calculated by using the equations LOD = 3xσ/ S and LOQ = 10xσ/S, where σ is the standard deviation of intercept, S is the slope. The LOD and LOQ were found to be 0.6589 μg/ml and 1.9772 μg/ml respectively of Cefixime Trihydrate and 0.6794 μg/ml and 2.0382 μg/ml of Ofloxacin.

Fig.8: Calibration curve for Cefixime Trihydrate at 287 nm

Table 3: Results of drug content and analytical recovery of Cefixime Trihydrate and Ofloxacin

Excess drug added to the analyte (%)	Drug	% Recovery		% RSD
		Method A	Method B	Method A	Method B
80	Cefixime Trihydrate	99.48	100.51	0.6570	0.6352
100		102.81	98.80	0.3589	0.8721
120		100.66	98.71	0.5873	0.6549
80	Ofloxacin	100.63	101.29	0.7487	0.5479
100		99.81	98.47	0.6589	0.6594
120		102.21	101.31	0.4470	0.7588

Table 4: Results of Intra-day and Inter-day Precision

Method	Drug	Intra-day Precision		Inter-day Precision
		SD	%RSD	SD	%RSD
A	Cefixime Trihydrate	0.6910	0.5821	0.6783	0.3214
B		0.7048	0.4961	0.6691	0.1962
A	Ofloxacin	0.7691	0.8367	0.1013	0.1029
B		0.8793	0.6658	0.1852	0.1297

Fig.9: Calibration curve for Ofloxacin at 296 nm

Table 2: Optical Characteristics and Precision

Sr. No.	Parameter	Cefixime Trihydrate	Ofloxacin
1	λ range	200-400 nm	200-400nm
2	Regression Equation (y=mx+c)	Y=0.023x+ 0.0171	Y=0.0214x+ 0.0139
3	Measured wavelength	287 nm	296 nm
4	Linearity range	5-25µg/ml	5-25µg/ml
5	Slope	0.023	0.0214
6	Intercept	0.0171	0.0139
7	Correlation coefficient (R^2)	0.9961	0.9973
8	Limit of Detection (LOD) µg/ml	0.6589	0.6794
9	Limit of Quantitation (LOQ)µg/ml	1.9772	2.0382

4. RESULTS AND DISCUSSION:-

The methods discussed in the present work provide a convenient and accurate way for analysis of Cefixime Trihydrateand Ofloxacin in its bulk and pharmaceutical dosage form. Absorbance maxima of Cefixime Trihydrate at 287nm and Ofloxacin at 296nm were selected for the analysis. Linearity for detector response was observed in the concentration range of 5-25 μg/ml for Cefixime Trihydrate and 5-25 μg/ml for Ofloxacin. Percent amount found for Cefixime Trihydrateand Ofloxacinin tablet analysis was found in the range of 101.36%, 98.54 and 100.59, 98.71 %respectively [Table 1]. Standard deviation and coefficient of variance for three determinations of tablet formulation, was found to be less than ± 2.0 indicating the precision of the methods. Accuracy of proposed methods was ascertained by recovery studies and the results are expressed as %recovery. % recovery for Cefixime Trihydrate and Ofloxacin was found in the range of 99.48 % and 100.63 % values of standard deviation and coefficient of variation was satisfactorily low indicating the accuracy of all the methods.% RSD for Intraday assay precision for Cefixime Trihydrate was found to be 0.5821 and 0.4961 for Method A and B, and for Ofloxacin, 0.8367 and 0.6658 for Method A and B. Interday assay precision for Cefixime Trihydrate was found to be 0.3214 and 0.1962 for Method A and B and for Ofloxacin 0.1029 and 0.1297 for Method A and B.The LOD and LOQ were found to be 0.6589 μg/ml and 1.9772 μg/ml respectively of Cefixime Trihydrate and 0.6794 μg/ml and 2.0382 μg/ml of Ofloxacin. Based on the results obtained, it is found that the proposed methods are accurate, precise, reproducible and economical and can be employed for routine quality control of Cefixime Trihydrate and Ofloxacin in bulk drug and its pharmaceutical dosage form.

5. CONCLUSION:

UV spectrophotometric methods for Cefixime Trihydrate and Ofloxacin were developed separately in bulk and tablet dosage form by, Absorbance maxima method and Area under curve method. Further, UV Spectrophotometric methods for the simultaneous estimation of Cefixime Trihydrate and Ofloxacin were in bulk and combined dosage form. The methods were validated as per ICH guidelines. The standard deviation and % RSD calculated for these methods are <2, indicating high degree of precision of the methods. The results of the recovery studies showed the high degree of accuracy of these methods. In conclusion, the developed methods are accurate, precise and selective and can be employed successfully for the estimation of Cefixime Trihydrate and Ofloxacin in bulk and pharmaceutical dosage form.

6. ACKNOWLEDGEMENT:

The authors are highly thankful to the Sahyadri College of Pharmacy, Methwade, Sangola, Solapur, Maharashtra, India for proving all the facilities to carry out the research work successfully.

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Received on 22.03.2016 Accepted on 15.04.2016

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Asian J. Pharm. Res. 2016; 6(2): 100-106

DOI: 10.5958/2231-5691.2016.00017.4