Simultaneous determination of moxifloxacin and cefixime by first and ratio first derivative ultraviolet spectrophotometry
© Attimarad et al.; licensee Chemistry Central Ltd. 2012
Received: 16 June 2012
Accepted: 3 September 2012
Published: 20 September 2012
The new combination of moxifloxacin HCl and cefixime trihydrate is approved for the treatment of lower respiratory tract infections in adults. At initial formulation development and screening stage a fast and reliable method for the dissolution and release testing of moxifloxacin and cefixime were highly desirable. The zero order overlaid UV spectra of moxifloxacin and cefixime showed >90% overlapping. Hence, simple, accurate precise and validated two derivative spectrophotometric methods have been developed for the determination of moxifloxacin and cefixime.
In the first derivative spectrophotometric method varying concentration of moxifloxacin and cefixime were prepared and scanned in the range of 200 to 400 nm and first derivative spectra were calculated (n = 1). The zero crossing wavelengths 287 nm and 317.9 nm were selected for determination of moxifloxacin and cefixime, respectively. In the second method the first derivative of ratio spectra was calculated and used for the determination of moxifloxacin and cefixime by measuring the peak intensity at 359.3 nm and 269.6 nm respectively.
Calibration graphs were established in the range of 1–16 μg /mL and 1–15 μg /mL for both the drugs by first and ratio first derivative spectroscopic methods respectively with good correlation coefficients. Average accuracy of assay of moxifloxacin and cefixime were found to be 100.68% and 98 93%, respectively. Relative standard deviations of both inter and intraday assays were less than 1.8%. Moreover, recovery of moxifloxacin and cefixime was more than 98.7% and 99.1%, respectively.
The described derivative spectrophotometric methods are simple, rapid, accurate, precise and excellent alternative to sophisticated chromatographic techniques. Hence, the proposed methods can be used for the quality control of the cited drugs and can be extended for routine analysis of the drugs in formulations.
Ultraviolet (UV) – visible spectroscopic method of analysis is widely used in the analysis of drugs in pharmaceutical formulations and for dissolution and disintegration studies due to its good sensitivity and cost effectiveness. In last three decades, derivative spectrophotometry has been extensively used in the determination of drugs in multi components having overlapping spectra, which eliminates interference from formulation matrix by using the zero-crossing techniques [1–4]. Ratio-spectra derivative spectrophotometric method is another useful technique for the estimation of drugs in their mixtures [5–8].
The derivative spectra methods allows us to use the wavelength of highest value of maxima or minima signals. Moreover, the presence of a more number of maxima and minima wavelengths gives an opportunity to select a particular wavelength for determination of active compounds without the interference from other compounds or formulation excipients. The derivative spectra methods are developed for simultaneous determination of moxifloxacin Hydrochloride (MOX) and cefixime trihydrate (CEF), a newly FDA approved multicomponent formulation for combination therapy.
Moxifloxacin (1-cyclopropyl-7-(S, S)-2, 8-diazabicyclo (4.3.0)-non-8-yl-6-fluoro-8-methoxy-1,4-dihydro-4-oxo-3-quinoline carboxylic acid hydrochloride, MOX, Scheme 1) is antimicrobial agent, it is fourth generation fluoroquinolone antibiotic. The mechanism of action involve inhibition of an enzyme topoisomerase II (DNA gyrase), which is essential for bacterial DNA replication . Several analytical methods have been reported for the determination of MOX in formulations and biological fluids, such as UV spectroscopic methods [23, 24], Spectrofluorometry , RP-HPLC [26–30], and capillary electrophoresis [31, 32].
The new combination of MOX and CEF is approved by Central Drugs Standard Control Organization (CDSCO) India for the treatment of lower respiratory tract infections in adults. Simultaneous determination of these drugs is essential in each step of initial formulation development and screening stage of any solid dosage form. A fast and reliable method for the dissolution and release testing of MOX and CEF were highly desirable. However, there are no simple and rapid analytical methods to estimate the drug content in the combined forms. Hence there is an urgent demand to develop a simple and rapid method such as spectroscopic method to assess the drug content in this combination. However, the major concern for these two drugs is the overlapping of absorption bands, which also restricts the direct measurement using UV spectroscopy. The objective of the current study was to develop rapid, accurate, reproducible, validated and economical first derivative and ratio first derivative analytical methods for the simultaneous determination of MOX and CEF from pure and in presence of formulation excipients.
Instruments and chemicals
UV-visible spectrophotometer (Shimadzu, 1700) with UV-probe software connected to computer was used for the drug estimation. Quartz cuvettes (1 cm) were matched and used for all absorbance measurements. Double distilled de-ionized water and Whatmann filter paper (no.41) were used throughout the experimental work. The sample of moxifloxacin certified to contain 99.81% was procured from Micro Labs, India and Cefixime trihydrate certified to contain 99.84% from Dr. Reddy's laboratory, India. Moxicip tablets containing 400 mg of moxifloxacin and suprax tablets containing 400 mg of cefixime were purchased from pharmacy.
Preparation of standard stock solution
Standard stock solutions of MOX and CEF (1 mg/mL) were prepared by dissolving 100 mg of drugs in water, separately. The working standard solutions of the respective drugs were prepared by serial dilution using water.
Method-D1 (First derivative Spectrophotometric method)
In the first order derivative method, aliquots of MOX and CEF standard stock solutions were accurately transferred in to 10 mL volumetric flasks, separately and volumes were completed with water. All the solutions were scanned from 200 to 400 nm in the spectrum mode. Thus obtained absorption spectra were derivatized from first to fourth order. First order derivative (n = 1) spectra showed good sensitivity and linearity hence the zero crossing wavelengths, 287 nm and 317.9 nm of first order derivative spectra were selected for analysis of MOX and CEF, respectively. The calibration curves were constructed and the concentration of individual drug present in the mixture was determined against calibration curve in quantitation mode.
Method-RD1 (First derivative of the ratio spectra)
Previously scanned absorption spectra of MOX solutions prepared at different concentrations (1 to 15 μg/mL) in its binary mixture with CEF was divided by the spectrum of the standard solution of CEF (10 μg /mL in water) to get the ratio spectra of MOX. The first derivative of the ratio spectra were than calculated. The amount of MOX was determined by measuring the first derivative signal at 359.3 nm. A similar procedure was followed for different concentrations of CEF (1 to 15 μg /mL ) with MOX and for division spectrum of the standard solution of MOX (8 μg /mL in water) was used. Similarly, content of CEF was determined by measuring the first derivative signal at 269.6 nm.
Assay of laboratory-prepared mixtures
The absorption spectrum was recorded for the laboratory prepared mixtures, against water as a blank. The intensity of the first derivative spectra of the laboratory prepared mixtures containing different ratios of MOX and CEF were measured at 287 nm and 317.9 nm respectively. The previously scanned zero order absorption spectra for the laboratory prepared mixture were divided by the spectrum of CEF (10 μg/mL) and by spectrum of MOX (8 μg/mL) separately for the determination of MOX and CEF respectively. The concentrations of MOX and CEF were calculated from their corresponding regression equations measuring the intensity of signals at 359.3 nm and 269.6 nm respectively.
Analysis of MOX and CEF in presence of tablet excipients
To determine the content of MOX and CEF simultaneously in the presence of tablet excipients 20 tablets of MOX 400 mg and 20 tablets of CEF 400 mg were weighed separately and average weight was determined. All the 40 tablets were powdered and correct amount of powder (equivalent to 20 mg of both the drugs) was dissolved in water using sonicater (30 min). After filtration, an appropriate aliquots were subjected to above methods and the amount of MOX and CEF were determined.
Validation of the methods
Newly developed methods were validated for linearity, accuracy, precision, limits of quantization and selectivity according to the ICH guidance.
For linearity six different concentration of MOX and CEF in the range of 1–16 μg/mL were analyzed. The limit of detection (LOD) and limit of quantification (LOQ) were determined by 3.3 σ/s and 10 σ/s criteria, respectively; where σ is the standard deviation of the analytical signal and s is the slope of the corresponding calibration curve.
Accuracy of the method was determined by calculating recoveries of MOX and CEF by standard addition method, in which pre-analyzed samples were taken (5 μg/mL ) and standard drug was added at three different levels i.e. 80%, 100% and 120%. The total amount of MOX and CEF were estimated by using the proposed methods in triplicate. The% recovery of the added pure drugs were calculated as% recovery = ((Dt-Ds)/Da) X 100, where Dt is the total drug concentration measured after standard addition; Ds drug concentration in the formulation mixture; Da drug concentration added.
Intraday and interday precision of the proposed methods were determined by estimating the MOX and CEF three times on the same day to obtain the repeatability and on three different days to obtain the reproducibility. Each assay was carried out on different samples of MOX and CEF. From the obtained data percent relative standard deviation (% RSD) was calculated.
Stability of the analytical solutions
This was evaluated by measuring absorbance of freshly prepared standard and sample solutions, and repeating the measurement after 24 h.
Results and discussion
Method development and validity
From the zero-order spectra first to four orders derivative spectra were obtained using digital differentiation for both the drugs. First order derivative spectra of both the drugs showed well-defined zones for determination of each analyte with a good sensitivity and linearity. Spectra with higher order of derivation had lower sensitivity and linearity, hence, only first order derivative spectra were selected for quantitative analysis. For first derivative spectroscopy, the spectra of the MOX and CEF obtained by scanning in water, were changed to first derivative spectra. For the first derivative spectra, different wavelengths (2, 4, 8 and 10 nm) were attempted to obtain the optimum Δλ. The results signified that 4 nm is an optimum Δλ and this wavelength was selected and used.
Validation parameters for first and ratio first derivative spectroscopic methods
Beer’s law range (μg /mL )
Intraday precision (%RSD)
Relatively very low % RSD for inter and intraday variations for both drugs in both methods confirms the precision of the methods (Table 1). RSD values found were well with the acceptable range indicating that these methods have excellent repeatability and reproducibility in the current experimental condition.
Amount of Base concentration (μg /mL )*
Amount of Pure Drug Added (μg /mL )
± RSD (%)
Estimation in pure and in presence of tablet excipients
Results of Assay of formulation by first and ratio first derivative spectroscopic methods
Weight taken (mg each)
% Amount found
± RSD (%)
± RSD (%)
Since information of placebo composition is not available, the comparative study was carried out for laboratory mixed standards and the formulation under investigation. When comparing the results (Table 3) of laboratory mixed and the formulation preparation one can conclude that the method is specific for the analyte used and the coexisting constituents do not affect the measurements.
Stability of the analytical solutions
Deviation from the mean initial absorbance is less than 2.4% for both MOX and CEF, which is well within the acceptable range (not more than ± 3%) indicating the stability of the analytical solutions.
A simple, rapid, accurate, and precise and nature friendly two derivative UV spectroscopic methods were developed for the simultaneous estimation of MOX and CEF in bulk drugs and in the presence of tablet excipients. The recovery studies suggested non-interference of formulations excipients in the estimation. Moreover, the present methods were rapid as compared to sophisticated chromatographic techniques, hence the proposed methods can be used for the quality control of the cited drugs and can be extended for routine analysis of the drugs in their pharmaceutical preparations.
Authors are thankful to Micro Labs, and Dr. Reddy's laboratory, India for proving gift samples of moxifloxacin and cefixime trihydrate, respectively. Author also wish their sincere thanks to College of Clinical Pharmacy, King Faisal University. KSA for providing necessary facilities for the work.
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