A simple and sensitive spectrophotometric method has been developed for the determination of sparfloxacin in bulk and pharmaceutical formulations, and in ...
Journal of Applied Spectroscopy, Vol. 77, No. 3, 2010
SPECTROPHOTOMETRIC DETERMINATION OF SPARFLOXACIN IN PHARMACEUTICAL FORMULATIONS AND URINE SAMPLES M. R. Jan, J. Shah,* and Inayatullah
UDC 543.42.062
A simple and sensitive spectrophotometric method has been developed for the determination of sparfloxacin in bulk and pharmaceutical formulations, and in artificial urine. Sparfloxacin was oxidized into a red colored product using ammonium monovanadate in acidic media. The proposed method was successfully applied to the determination of sparfloxacin in different pharmaceutical formulations (tablets) and in a spiked urine sample. The influence of commonly used excipients on the determination of sparfloxacin was studied. Percentage recoveries in the range of 98.0 ± 0.14 % to 100.0 ± 0.20 % were obtained. The observed data have been evaluated statistically which showed high accuracy and precision. Keywords: sparfloxacin, ammonium monovanadate, spectrophometry, pharmaceutical formulations. Introduction. Sparfloxacin [5-amino-1-cyclopropyl-7-(cis-3,5-dimethyl-1-piperazinyl)-6,8-difluoro-1,4-dihydro4-oxo-3-quinoline carboxylic acid] is a third-generation antibacterial used in the treatment of lung infection, urinary tract infection, and cutaneous allergy. Sparfloxacin (SPFX) is receiving attention due to its broad spectrum of activity against Gram negative bacteria and improved activity against Gram positive bacteria, including anaerobe pathogens, its potency, and excellent pharmacokinetic profiles [1–3]. Different analytical methods have been developed for quantitative determination of sparfloxacin in pharmaceutical preparations like nonaqueous titration [4], HPLC [5–7], DC polarography [1], and capillary zone electrophoresis [3, 8, 9]. The spectrophotometric methods available in the literature for the determination of sparfloxacin suffer from problems in one way or another. The method reported by El-Didamony [10], which is based on ternary complexes of SPFX with Pd (II) and eosin, has been applied neither to the interference effects of excipients nor to biological samples. The colored product of the reaction between sparfloxacin and ammonium reineckate is a precipitate of an ionpair complex whose separation and redissolution in acetone are required before the determination [11]. Both the adverse effects of excipients and the biological samples also have not been investigated. The spectrophotometric methods based on the ion-pair complex formation with bromothymol blue (BTB) and methyl orange (MO) reported by Marona et al. [2] and Patel et al. [12] respectively suffer from disadvantages like the extraction process, the use of organic solvents for the extraction, and a narrow range of the Beer’s law determination. The charge transfer reaction between the alizarine red and sparfloxacin [13] also shows low sensitivity. Askal et al. [14] used N-bromosuccinimide as a reagent for the indirect determination of sparfloxacin. The method is tedious, requires a long time for the completion of the reaction, and has low sensitivity. The present work was carried out to develop a sensitive and direct spectrophotometric method for the determination of sparfloxacin based on its oxidation by ammonium monovanadate. The resultant red colored product was monitored at 530 nm. Experimental. Instruments. A spectrophotometer (SP-300 Optima Inc., Tokyo, Japan) with 1 cm matched cells and an electric thermostatic water bath (MC 02810175 Yu Jia China) were used.
∗
To whom correspondence should be addressed.
Institute of Chemical Sciences, University of Peshawar, N.W.F.P., Pakistan; e-mail: jasminshah2001@ yahoo.com. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 77, No. 3, pp. 432–437, May–June, 2010. Original article submitted August 27, 2009. 400
0021-9037/10/7703-0400 ©2010 Springer Science+Business Media, Inc.
Reagents. All reagents used were of high grade purity: ammonium monovanadate (reagent grade, ACS, Scharlau), sulfuric acid (Merck, 95–98%), and methanol (Merck). Standard reference sparfloxacin was gifted by Libra Pharmaceuticals (Pvt) Ltd., Peshawar, Pakistan. Commercial formulations of sparfloxacin (Sparxin, Sparcin, and Quspar) were purchased from the local market. Solutions. 1. Ammonium monovanadate solution (0.04M). Prepared by dissolving 1.148 g of ammonium monovanadate in sufficient hot distilled water, transferring it to a 250 ml volumetric flask, and making volume up to the mark with distilled water. 2. Sulfuric acid solution (7M). Prepared by adding 95.28 ml of 95–98% pure sulfuric acid to sufficient cold distilled water and making the volume up to the mark with cold distilled water in a 250 ml volumetric flask. 3. Standard sparfloxacin solution (1000 g/ml). Prepared by dissolving 0.1 g of authentic standard sparfloxacin in 50 ml of methanol with vigorous shaking and making the volume up to the mark with methanol in a 100 ml volumetric flask. A 200 μg/ml working standard solution was prepared from the stock solution by diluting with distilled water. 4. Procedure for dosage form. Five tablets were weighed, ground, and mixed. After fine powdering of the tablets, an accurately weighed portion of the sample containing the equivalent of 100 mg of sparfloxacin was dissolved in 50 ml of methanol and vigorously shaken. The solution was filtered and diluted with distilled water in a 100 mL flask. General Procedure. Sparfloxacin solutions in the range of 4–20 μg/ml from the 200 μg/ml working standard solution were transferred to 100 ml beakers. To each solution, 5 ml of the ammonium monovanadate (0.04 M) solution was added, followed by the addition of 5 ml of sulfuric acid (7 M). The mixture was heated on a boiling water bath for 30 s. On cooling, the contents of the beakers were transferred to a 25 ml volumetric flask and diluted up to the mark with distilled water. The absorbance of the resultant red colored product was measured at 530 nm by Optima SP-300 spectrometer against a reagent blank prepared by the same method without adding the drug. Procedure for spiked artificial urine. An aliquot of the methanolic solution of sparfloxacin containing 1, 5, and 10 μg/ml was added to 10 ml of an artificial urine sample [15] and shaken well. Then we proceeded as described under general procedure, and the content of sparfloxacin was determined from a calibration plot. A blank value was determined by treating sparfloxacin-free artificial urine in the same way. Results and Discussion. The spectrophotometric method for the determination of sparfloxacin is based on the oxidation of sparfloxacin with ammonium monovanadate in an acidic medium. The red colored oxidized product of sparfloxacin shows a maximum absorption at 530 nm (Fig. 1). Optimization of conditions. For the maximum oxidized product formation, the effects of acidity, temperature, heating time at optimum temperature, and concentration of ammonium monovanadate have been studied. Effect of temperature and heating time. The reaction between sparfloxacin and ammonium monovanadate was relatively slow at room temperature. Therefore, the effects of temperature and heating time on the formation of oxio dized product of sparfloxacin reacted with ammonium monovanadate were studied in the ranges of 40–100 C and 30 s o to 5.0 min, respectively. The maximum absorbance was found at 100 C and at the heating time of 30 s (Fig. 2). Effect of acidity. The effect of acidity on the redox reaction was studied in the range of 1–10 M of the sulfuric acid solution. The maximum absorbance was obtained with 10 ml of the 7 M sulfuric acid solution. Above and below the optimum concentration of acid, a decrease in the absorbance was observed, therefore, 7 M sulfuric acid was further used for the oxidation of sparfloxacin with ammonium monovanadate (Fig. 3a). 2.4. Effect of concentration of ammonium monovanadate solution. The effect of the ammonium monovanadate solution concentration on the reduction-oxidation reaction with sparfloxacin was studied in the range of 0.01 M to 0.06 M. It was observed that with increase in concentration from 0.01 M to 0.04 M the absorbance increased, after which it remained constant up to 0.06M (Fig. 3b). The effect of the volume of ammonium monovanadate solution (0.04 M) on the absorbance behavior of the colored product was also investigated. It was found that 15 ml of the ammonium monovanadate solution (0.04 M) gave the maximum absorbance of the product, which remained unchanged up to 20 ml. Therefore, 15 ml of the 0.04 M ammonium monovanadate solution was used throughout the analysis. Figures of merit. Under the optimum experimental conditions of the proposed method, a linear relation between the absorbance and the sparfloxacin concentration was observed (Fig. 4). Beer’s law was obeyed in a concentration range of 0.8–28 g/ml. The linear regression equations, slopes, intercepts, correlation coefficients, the standard 401
Fig. 1. Absorption spectra of red colored oxidized product of sparfloxacin.
Fig. 2. Effect of temperature (a) and heating time (b) on the formation of colored product from oxidation of sparfloxacin.
Fig. 3. Effect of acidity (H2SO4) (a) and ammonium monovanadate concentration (b) on the formation of colored product from oxidation of sparfloxacin.
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Fig. 4. Effect of concentration on the absorbance of the product of sparfloxacin oxidation. TABLE 1. Analytical Parameters for the Spectrophotometric Determination of Sparfloxacin
λmax, nm Beer,s law limits, μg/ml Molar absorptivity, l⋅mol–1⋅cm–1 Limit of detection, μg/ml Limit of quantification, μg/ml Regression equation Slope (b) Intercept (a) Correlation coefficient (r) Standard deviation, μg/ml Relative standard deviation, %
530 0.8–28 4⋅103 0.15 0.50 y = 0.009x + 0.002 0.009 0.002 0.9996 0.05 8.77
TABLE 2. Determination of Sparfloxacin (10 μg/ml) in the Presence of Excipients Excipient Glucose
Sucrose
Sorbitol
Lactose Starch Talc
Excipient added, mg
Recovery RSD, %
0.050 0.500 1.000 0.050 0.500 1.000 0.050 0.500 1.000 0.050 0.500 1.000 0.025 0.050 0.025 0.050
100 0.38 100 0.38 100 0.38 100 0.38 100 0.38 97 0.41 100 0.38 100 0.38 100 0.38 100 0.38 97 0.41 97 0.41 100 0.27 102 0.38 105 0.38 108 0.38 403
TABLE 3. Evaluation of Accuracy and Precision of the Proposed Method for Sparfloxacin Determination Sample
Amount taken, μg/ml
Amount found, μg/ml
Recovery RSD, %
Confidence limit
5.0 10 15 5.0 10 15 5.0 10 15
5.0 9.8 14.7 5.1 10 15 5.0 9.9 15
100 0.2 98 0.16 98 0.14 102 0.2 100 0.16 100 0.14 100 0.2 99 0.16 100 0.14
5.0 2.48⋅10–2 9.8 3.90⋅10–2 14.7 5.12⋅10–2 5.1 2.98⋅10–2 10 3.98⋅10–2 15.0 5.59⋅10–2 5.0 2.48⋅10–2 9.9 3.93⋅10–2 15.0 5.22⋅10–2
Sparcin tablets
Quspar tablets
Sparxin tablets
TABLE 4. Recovery Test for the Determination of Sparfloxacin in Tablets by the Proposed Method Pharmaceutical preparation Sparcin tablets (100 mg/tablet) Quspar tablets (100 mg/tablet) Sparxin tablets (100 mg/tablet)
Amount added,
Amount found, μg/ml
RE, %
Recovery RSD, %
5.0 10 15 5.0 10 15 5.0 10 15
5.0 9.8 14.7 4.9 9.8 15 4.9 9.9 15
0.0 2.0 2.0 2.0 2.0 0.0 2.0 1.0 0.0
100 0.20 98 0.16 98 0.14 98 0.20 98 0.16 100 0.14 98 0.20 99 0.16 100 0.14
TABLE 5. Application of the Proposed Method to the Determination of Sparfloxacin in Spiked Urine Sample Spiked amount, μg/ml
Amount found, μg/ml
Recovery RSD, %
1.0 5.0 10
1.0 4.9 9.8
100 0.33 98 0.34 98 0.82
Note. Each result is the average of separate triplicate analysis.
deviation, and the relative standard deviation of the response factors are given in Table 1. The molar absorptivity was 3 –1 –1 found to be 4.0⋅10 l⋅mol ⋅cm . The limit of detection (LOD) and limit of quantification (LOQ) were determined and found to be 0.15 and 0.50 μg/ml, respectively. Effect of interferences. To evaluate the selectivity of the developed method for the analysis of pharmaceutical preparations containing sparfloxacin, the interference effects of various excipients were studied. Solutions containing sparfloxacin and one of the excipients taken separately in concentrations five, ten, fifty, and a hundred times greater than that of sparfloxacin were analyzed by the proposed method. A level of interference was considered to be acceptable if the error was not higher than 3% relative to the expected sparfloxacin value. No interferences were observed 404
in the determination of sparfloxacin in the presence of the common excipients studied except talc (Table 2). The talc tolerable concentration is below 5 mg but within the formulation range. Precision and accuracy of the method. The precision and accuracy of the proposed method were checked by determining the sparfloxacin concentration in replicate for each concentration within the Beer’s law limits. The results are summarized in Table 3. The relative standard deviation (RSD) was considered very satisfactory, and the recovery test results were found to be in the range of 98–102%, indicating a good accuracy. The proposed method was found useful for routine and quality control analysis of sparfloxacin both in pharmaceutical preparations and in pure form. Analysis of pharmaceutical preparations. The proposed method has been successfully applied to the determination of sparfloxacin in a commercial preparation (tablets). The results show good recovery data and are in good agreement with the label claims (Table 4). Moreover, the validity of the proposed method with respect to dosage forms (three different brands of tablets) was tested for possible interference with a standard addition method. The recoveries obtained were in the range of 98–100% which shows the selectivity of the developed method towards sparfloxacin in commercial pharmaceutical preparations. Analysis of spiked urine sample. Good sensitivity obtained by the proposed method suggested the determination of sparfloxacin in urine samples. Sparfloxacin was orally administered at doses of 100 mg two times daily, which resulted in a urine level of concentration about 2–4 μg/ml. The obtained results shown in Table 5 are satisfactorily precise and accurate. Conclusion. A simple, precise, and accurate method was developed for the determination of sparfloxacin in pharmaceutical preparations and spiked urine samples. As compared to HPLC methods, the reagents used in the proposed spectrophotometric method are readily available and the procedure does not involve any tedious sample preparation. The developed method is readily adaptable to use in quality control laboratories.
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