Simultaneous Spectrophotometric Determination of Cefixime and Moxifloxacin in Bulk Drug and Drug Formulation by Absorption Ratio Method

About Authors:
Shah Chirag K.*, Umalkar Deepak, Dr. Rajesh KS.
Parul Institute of Pharmacy, Gujarat University,
Waghodia-391760, Dist. Vadodara,
Gujarat, India.
*cks2484@gmail.com

Abstract:
The present manuscript described the simple, rapid, accurate, sensitive, precise and economical Q- absorption ratio method for simultaneous determination of Cefixime and Moxifloxacin in combined tablet dosage form. Absorption ratio method uses the ratio of absorbances at selected wavelengths, one which is isoabsorptive point and other being the λ_max of the one of the components. Cefixime and Moxifloxacin shows isoabsorptive point at 275nm in 0.1N HCl. The second wavelength used is 295nm, which is λ_max of Moxifloxacin. The concentrations of the drugs were determined by using ratio of absorances at isoabsorptive point and at λ_max of Moxifloxacin. The method successfully applied to the Pharmaceutical formulation because no interference from the tablet formulation excipients was found. The results of analysis have been validated statistically and by recovery studies.

Reference Id: PHARMATUTOR-ART-1355

Introduction:
Cefixime Trihydrate (CEF), (6R,7R)-7-{[2-(2-amino-1,3-thiazol-4-yl)-2-(carboxy methoxyimino)acetyl]amino}-3-ethenyl-8-oxo-5-thia 1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic trihydrate (Figure 1), is third generation Cephelosporin antibiotic¹. Moxifloxacin (MOX), 1-cyclopropyl-7-[(1S,6S)-2,8-diazabicyclo[4.3.0]non-8-yl]-6-fluoro-8-methoxy-4-oxo-quinoline-3-carboxylic acid(Figure 2), is floroquinolone antibiotic². This combination is used for treatment of lower Respiration tract infection in adult³. Literature reveals Spectrophotometric⁴, HPLC⁸ methods for Cefixime in Pharmaceutical dosage forms and as well as biological fluids. Literature survey also reveals Spectrophotometric¹⁴ and HPLC¹⁸ methods for Moxifloxacin in Pharmaceutical dosage forms and as well as biological fluids. The combination is not official in any pharmacopeia; hence no official method is available for the estimation of Cefixime and Moxifloxacin in their combined dosage forms. Literature survey does not reveal any simple spectrophotometric or other method for simultaneous estimation of Cefixime and Moxifloxacin in combined dosage forms. The present work describes simple, sensitive, rapid, accurate and economical spectrophotometric method for simultaneous estimation of both the drugs in their combined tablet dosage forms.

Material and Method:
A Shimadzu model 1800 double beam UV/Visible Spectrophotometer with spectral width of 2nm, wavelength accuracy 0.5nm and a pair of 10nm matched quartz cell was used to measure absorbance of all the solutions. Spectra were obtained automatically from UV-Probe ver2.34 software.

Reagents and Materials:
Cefixime powder was gifted by Kaptab Pharmaceuticals, Vadodara, India. Moxifloxacin powder was gifted by BDR Pharmaceutical International Pvt. Ltd., Mumbai, India. HCl AR Grade was procured from SDFCL, Baroda, India.

Preparation of the standard stock solution:
An accurately weighed quantity of CEF (10 mg) and MOX (10 mg) were transferred to a separate 100 ml volumetric flask and dissolve and diluted to the mark with 0.1N HCl. To obtain standard solution having concentration of Cef (100 μg/ml) and Mox (100 μg/ml).

Methodology:
The Absorption Ratio method based on measurements at two wavelengths. Two dissimilar chromophores must necessary have different powers of light absorption at some points or in linear absorption. A set of two simultaneous equations obtained by using mean absorptivity values are given below. Seven working solution having concentration 2, 4, 6, 8, 10, 12 and 14 μg/ml of both the drug Cefixime and Moxifloxacin were prepared respectively in 0.1N HCl and the absorbance at 275nm (Isoabsorptive point) and 295nm(λ_max of Moxifloxacin) were measured and absorptivity coefficients were calculated using calibration curve. The absorbance and absorptivity at this wavelength were substituted in following equations to obtain the concentration of both drugs. From overlain spectra Fig3.

Cx = Qm - Qy  X (A₁/Ax₁) ……………(1)
         Qx - Qy  

Cy = Qm - Qx  X (A₁/Ay₁) ……………(2)
         Qy - Qx

Q_M, Q_X, and Q_Y were obtained as below:
Q_M = A₂/A₁, Q_X = ax₂/ax₁, Q_Y = ay₂/ay₁

Where, A₁ and A₂ were absorbance of sample at 275 nm and 295 nm respectively, ax₁ and ax₂ are absorptivity of Cefixime at 275 nm and 295 nm, ay₁ and ay₂ are absorptivity of Moxifloxacin at 275 nm and 295 nm. 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. Results of which are reported in Table.3

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Validation of the method
1.   Linearity and Range: The calibration curves were plotted over a concentration range 2-14 μg/ml for each Cef and Mox. Accurately measured standard stock solution of each Cef and Mox (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 ml) were transferred to a series of 10 ml volumetric flask separately and diluted upto the mark with 0.1N HCl. The absorbances of the solutions were then measured at 275 nm and 295 nm. The calibration curves were constructed by plotting absorbances V/S concentration and the regression equations were calculated which are shown in Fig4 and Fig5 respectively.
2.   Precision (Repeatability): Precision and accuracy was studied by covering three different concentrations {low (80%), medium (100%) and high (120%)} viz. 8, 10 and 12 μg/ml. Absorbance of the solutions was measured for three replicate samples of each concentration. Intra-day precision studies were run in triplicate in the same day and inter-day on three consecutive days. Precision data is shown in Table 1

Table 1: Precision Data

3.   Accuracy (% Recovery):
It is the measure of closeness between the actual value and the analytical value that is calculated by applying the test procedure for a number of times. Recovery was done at three different levels viz. 80%, 100% and 120%, within the beer’s limit for both the drugs. The previously analyzed sample of concentration 5 μg/ml was spiked with known concentrations of the pure samples and then reanalyzed using the proposed methods. Percentage recovery was calculated using the equations for both the methods. Percentage recovery is given in Table 2.

4.   Limit of detection and quantification:
Limit of detection (LOD) is the minimum concentration of the analyte in the sample which can be analyzed by the instrument. Limit of quantification (LOQ) is the minimum concentration of the analyte that can be reliably quantified. Equations for the LOD and LOQ were listed below. The values of LOD and LOQ are given in Table 3.

LOD = 3 SD / SLOP          ………… (3)

LOQ=  10 SD / SLOP         ………… (4)

5.   APPLICATION OF THE DEVELOPED METHOD ON TABLET DOSAGE FORM:
20 tablets were weighed, crushed and an accurately weighed sample equivalent to 20mg of Cefixime and 20mg of Moxifloxacin, and the stock solution of this was prepared in 0.1N HCl, sonicated for 10 min, was then filtered through Whatman filter paper and then volume was made up to 50 ml with methanol. This stock solution contains 400 mg/ml of each drug. Then the appropriate dilutions were made using 0.1N HCl, in the linearity range as previously mentioned. All determinations were carried out three times. The absorbance of the prepared solutions was observed at 275 nm and 295 nm and then the concentration of both the drugs was calculated using equation 1 and 2.

Results and Discussion:
In absorption ratio method (Q-Analysis), the primary requirement for developing a method for analysis is that the entire spectra should follow the Beer’s law at all the wavelength, which was fulfilled by both these drugs. The two wavelengths were used for analysis of the drugs were 275 nm (iso-absorptive point) and 295 nm (λ_max of Moxifloxacin) at which the calibration curves were prepared for both the drugs. The UV overlay spectra of Cef (285 nm) and Mox (295 nm) showing isoabsorptive point (275 nm) in 0.1N HCl is shown in figure 3.

The validation parameters were studied at all the wavelengths for proposed method. Accuracy was determined by calculating recovery, and the mean was determined (Table 2.). The method successfully used to determine the amounts of Cef and Mox in present tablet dosage forms. The results obtained were in good agreement with the corresponding labeled amount (Table 3). Precision was calculated as repeatability and intra and inter day variation (%RSD) for both the drugs. Optical characteristics and summary of the validation parameters for the method is given in Table 3. By observing the validation parameters, the method was found to be simple, sensitive, accurate, precise and economical. Hence the method can be employed for the routine analysis of these two drugs in combined dosage form.

Table3. Result Table

LOD= Limit of Detection, LOQ= Limit of Qualification, CEF= Cefixime, MOX= Moxifloxacin

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Acknowledgment:
The author is thankful to Kaptab Pharmaceutical, Vadodara, India. for providing the Cefixime and also to BDR Pharmaceutical International Pvt. Ltd., Mumbai, India. for providing the Moxifloxacin for research work. The authors are highly thankful to Parul Institute of Pharmacy, Gujarat University, Waghodiya, Dist. Vadodara, Gujarat, India. for providing all the faculties to carry out the work. The author also thankful to the Mr. Stavan Master, lecturer, Baroda College of Pharmacy, Gujarat University, Waghodiya, Dist. Vadodara, Gujarat, India. sharing his knowledge with me.

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