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IJRPC 2014, 4(4), 972-976 M Phatak et al. ISSN: 22312781

INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY
Available online at

Research Article
DEVELOPMENT AND VALIDATION OF A HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS
QUANTIFICATION OF ALBENDAZOLE AND CLOSANTEL FROM
VETERINARY FORMULATION
M. SPhatak*, VV. Vaidya and H. MPhatak
Department of Chemistry, Ramnarain Ruia College, Matunga, Mumbai-400019, India. ABSTRACT
A simple and rapid Reverse Phase HPLC method has been developed for the simultaneous quantification of Closantel and Albendazole from veterinary anthelmintic formulation. HPLC analysis was performed on C18 column maintained at 30ºC using a simple mixture of acetonitrile, distilled water and methanol as isocratic mobile phase at a flow rate of 1.8ml per minute at detection wavelength of 254nm. The method was validated for accuracy, precision, linearity, specificity and sensitivity in accordance with International Conference on Harmonization guidelines. Good linear correlation coefficients (r2>0.999) were obtained for calibration plots in the range of 50–150μg/ml for Closantel and 25 – 75 μg/ml Albendazole. Intraday and Interdayprecision of retention times and peak areas were less than 2.0%. Accuracy of the method was between 99.35% and 99.86% for Closantel and 99.58% and 100.16% for Albendazole. Validation revealed the method to be specific, accurate, precise, reliable and reproducible. The method was successfully used for quantitative analysis of these analytes from marketedVeterinary anthelmintic formulation.
Keywords: Simultaneous determination; RP-HPLC; Albendazole; Closantel; veterinary formulation.
analytical methods used were LC MS/MS and anthelmintic agent (Figure 1). It is a broad- Albendazole is a benzimidazole class of drug spectrum antiparasitic agent used against being used in the treatment for parasitic several species and developmental stages of infections (Figure 2). It is a broad spectrum trematodes, nematodes and arthropods. The anthelmintic effective against roundworms, anti-trematode activity of closantel is mainly tapeworms, and flukes of domestic animals used against liver fluke. The anti-nematode and humans4. Albendazole is available and anti-arthropod activity is especially used commercially in the form of tablets for human against those species which feed on blood or use and tablets and oral drench for veterinary plasma. Closantel has shown beneficial effect use. Albendazole is used alone or in when used in humans for the treatment of combination with other anthelmintic drugs for better efficacy. Various analytical method have Closantel is available commercially as oral treatments in the form of tablets and drench Albendazole5,6,7. formulation alone or in combination with other There is no method available currently for the anthelmintic agents for veterinary use. There determination of Closantel and Albendazole are a few methods available for the from a veterinary formulation. In the present quantification of Closantel from biological work the development and validation of a matrix2 or pharmaceutical presentation3. The simple and rapid method for the simultaneous IJRPC 2014, 4(4), 972-976 M Phatak et al. ISSN: 22312781

quantification the two drugs from an oral
Calibration Curve
drench formulation. Standard solutions of 50 - 150 μg/mL of Closantel and 25-75 μg/mL of Albendazole MATERIALS AND METHODS
were analyzed to check the linearity range. Instruments
Specificity
Technologies 1200 HPLC model with photo The specificity of the method was ascertained diode array detector, Rheodyne injector having by analysing the standards and the samples. 20μL loop volume. Separation was carried out The peaks of Closantel and Albendazole in using Thermo Electron Corporation, Hypersil sample were confirmed by comparing the BDS C18 (150 x 4.6 mm, 5μ) column. retention time and spectra of the standards8. Detection was carried out using UV-visible detector. The flow rate was kept at 1.8 ml/ min Precision
and the column oven was maintained at 30 ºC. Precision was evaluated by injecting six The total chromatography run time was 4 injections at the working level concentrations of Closantel (100μg/mL) and Albendazole (50μg/mL) were analysed to examine the Reagents
precision of the method. Intraday precision All chemicals and solvents used were of HPLC and interday precision for the developed grade. Distilled water generated from TQA methods were measured in terms of %RSD. The % RSD of the six replicates was Closanteland Albendazole were provided as considered for intraday precision and % RSD gift sample by Cipla Ltd., India. Veterinary of six replicate injections, injected on two anthelmintic formulation Closal drenchwas different days were considered for inter day purchased from local market. precision. The concentration values for both intraday precision and interday precision were Standard Solutions
calculated and percent relative standard Stock solutions, of 1 mg/ml of Closantel and deviation were calculated using following 0.5 mg/ml of Albendazole were freshly prepared individually in methanol. Aliquots of Closantel (50-150μg/mL) and Albendazole % RSD = [S/X] 100,
(25-75 μg/mL) were prepared by subsequent dilutions of the stock solutions in the mobile S is standard deviation and X is mean of the sample analyzed. Sample Preparation
Accuracy
The Closal drench is available as a formulation Accuracy of the method was determined by containing 19 g/L of Albendazole and 37.5 g/L recovery experiments. Recovery experiments of Closantel. The sample for analysis was were carried out by the standard addition prepared bydiluting 1.1 ml of the formulation to method. This study was performed by diluting 100 ml with mobile phase. Further, 5 ml of this adding 10 ml, 10 ml and 20 ml each of the of solution is diluted to 20 ml to give the sample standard solution of Closantel (1000 μg/ml) and Albendazole (500 μg/ml) to three different volumetric flasks containing the 1.1 ml of sample solution has a concentration of 103.1 sample. The resultant sample solutions were correspondingly diluted to provide 50%, 100% and 150% of Closantel and Albendazole concentration compared to the standard Preparation of Mobile Phase
solution. The amounts of standard recovered The mobile phase is prepared by mixing were calculated in terms of mean recovery acetonitrile, distilled water and methanol. In a with the upper and lower limits of percentage volumetric cylinder 600 ml of acetonitrile is relative standard deviation8. taken to which 300 ml of distilled water and 100 ml of methanol is added. The mobile RESULTS AND DISCUSSION
phase is sonicated for 2 minutes and filtered Optimization of the method was carried out through 0.45 μm Teflon filter before using in the HPLC system. acetonitrile,distilled water and methanol. A solvent combination of acetonitrile: D.W.: methanol (60:30:10) gave a satisfactory separation of the Closantel and Albendazole. This optimized mobile phase separated


IJRPC 2014, 4(4), 972-976 M Phatak et al. ISSN: 22312781

Albendazole at 1.47 min and Closantel at 2.21
solution is shown in figure 5. Even though min respectively. The column efficiency, Closantel and Albendazole show common resolution and peak asymmetry and resolution therapeutic activities, they are entirely different were calculated for the standard solutions and in their chemical nature. Good resolution of are presented in Table 1.Increasing the Closantel and Albendazole in a simple concentration of distilled water in the solvent isocratic mobile phase were achieved. The system resulted in deterioration of the peak shape and retention of peaks on the column, sample purchased were done and the results resulting is longer analysis time. On the other demonstrated consistency between different hand increasing the organic phase caused the samples and analysis conducted on different Closantel peak to elute very close to the placebo peaks. The calibration curves of Closantel and Albendazole were linear in the CONCLUSION
range of 50- 150μg/mL for Closantel and 25- Based on the above results it can be 75 μg/mL for Albendazole. The linearity concluded that the developed method can be experiment observations and the slope, y- used for the simultaneous quantification of intercept and regression coefficient (r2) are Albendazole and Closantel for routine quality shown in Table 2. control analysis. Accuracy rom sample matrices was between 99.52% &100.10% and 99.67% and 99.98% respectively for Closantel and Albendazole. We would like to thank Cipla Ltd., India for The individual standard chromatogram for providing the analytical standards for the Closantel and Albendazoleare shown in figure 3 and 4, while the chromatogram of sample Fig. 1: Structure of Closantel
Fig. 2: Structure of Albendazole
IJRPC 2014, 4(4), 972-976 M Phatak et al. ISSN: 22312781

Fig. 3: Typical chromatogram for Closantel
Fig. 4: Typical chromatogram for Albendazole
Fig. 5: Typical chromatogram for sample solution
IJRPC 2014, 4(4), 972-976 M Phatak et al. ISSN: 22312781

Table 1: Chromatographic peak characteristics
Parameter
Albendazole
Closantel
Theoretical plates Table 2: Linearity experiment observations
Albendazole
Closantel
Linearity level
Peak area
Peak area


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