ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2009, том 64, № 2, с. 187-191
^=ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
DETERMINATION OF GLIMEPIRIDE IN PHARMACEUTICAL FORMULATIONS USING HPLC AND FIRST-DERIVATIVE SPECTROPHOTOMETRIC METHODS
© 2009 I. U. Khan, F. Aslam, M. Ashfaq, M. N. Asghar
Department of Chemistry, Faculty of Science, Government College University Lahore-54000, Pakistan Received 17.05.2007; in final form 20.11.2007
Two validated analytical methods have been developed to determine glimepiride in pharmaceutical formulations using HPLC and 1st order derivative spectrophotometry techniques. Employing reverse phase HPLC method, the drug was analyzed by pumping a mixture of acetonitrile and 2% formic acid solution, pH 3.5 (80 : 20 v/v) through a C18 column (250 x 4.6mm, 5 |im) and detecting the eluents at 228 nm. The linearity range was found to be 20-140 pg/mL with mean recovery of 100.52 ± 0.33%. The second method was based on the formation of a complex of the drug with 2,3,5-triphenyl-2H- tetrazolium chloride in basic media. 1st order derivative spectrum made it possible to detect the complex at 413.5 nm. The linearity range was found to be 40-160 |g/mL, with mean recovery of 100.33 ± 0.47%. Both the proposed methods can reliably be used for routine analysis of glimepiride in raw material as well as in pharmaceutical formulations.
Glimepiride, 3 -ethyl-2,5-dihydro-4-methyl-4-[2-[4-[[[[(trans-4-methylcyclohexyl) amino] carboxyl]ami-no] sulfonyl] phenyl] ethyl]-2-oxo-1-H-pyrrol-1-carbox-amibe] belongs to 2nd generation sulphonylurea which is being used for the treatment of non-insulin dependant diabetes mellitus (NIDDM), in order to achieve appropriate control of blood glucose level [1]. In addition, it maintains a better physiological regulation of insulin secretion than other sulphonylurea during physical exercise [2]. Multiple analytical procedures have been reported for in vivo and in vitro determination of the drug and its metabolites using LC-MS-MS [3-5], HPLC (6-7), HPLC after pre-column derivatization [8], and derivative uv-specttrophotometric method [9]. Kovarikova et al. [10] reported HPLC method for the determination of glimepiride under deliberate stress conditions, however, the retention time of more than 20 min hinders its application to routine analysis.
In the present studies, we report two new analytical methods, one is based on HPLC and the other on derivative spectrophotometry, for the determination of glime-piride in pharmaceutical formulations. The HPLC method utilizes C18 column and a salt-free mobile phase with retention time reduced to only seven minutes. The derivative spectrophotometric method is based on the formation of a complex of the drug with 2,3,5-ttriphenyl-2H-tetrazolium chloride, showing the absorption maximum at 413.5 nm. This reaction is highly selective for glime-piride and does not involve any interference by the ex-cepients. These methods showed comparable results and could equally be applied to routine quality control analysis of the drug.
EXPERIMENTAL
Chemicals and reagents. All the solvents used were of highest purity grade. HPLC grade acetonitrile (Merck) was used for making mobile phase. Sodium hydroxide, 2,3,5-triphenyl-2H- tetrazolium chloride and formic acid were of analytical reagent grade (Merck). Glimepiride reference substance (assigned purity 99.78%) was kindly donated by Schazoo Laboratories Pvt. Ltd. (Pakistan), while pharmaceutical glimepiride was purchased from local market. Glimepiride tablets available at the market were claimed to contain 1, 2, 3 and 4 mg of the active component. We used tablets containing 4 mg glimepiride (Glemex, 4 mg) for our experiment. Double distilled water was used throughout the experiment.
Conditions. The HPLC analysis was performed on Shimadzu LC-10A HPLC system (Kyoto, Japan) which was equipped with an LC 10 ADvp pump, a Rheodyne injection valve with a 20 ^L loop and a UV detector. The detector wavelength was set at 228 nm. Routine analysis were carried out isocratically using Varian C18 column (256 x 4.6 mm I.D., 5 ^m particle size) and mobile phase consisting of acetonitrile and 2% formic acid solution, pH 3.5 (80 : 20 v/v) with a flow rate of 0.5 mL/min.
Spectrophotometric analysis was performed on CECIL CE-7200 UV-Visible Spectrophotometer (England) using 10 mm quartz cells. The first-derivative spectra of the sample solutions were recorded at 413.5 nm using fixed slit width of 2 nm over the wavelength range of 350-600 nm at room temperature.
Preparation of mobile phase, stock and working standard solutions for HPLC. Mobile phase consisting of acetonitrile and 2% formic acid solution (pH 3.5)
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was prepared by mixing the two solutions, 80 : 20 (v/v). pH of formic acid solution was adjusted to 3.5 with 0.1 N Sodium hydroxide before mixing with acetoni-trile. The solution was degassed by sonication before use. Stock solution of glimepiride reference substance (1 mg/mL) was prepared by dissolving 0.1g glimepiride reference in a small volume of mobile phase in 100 mL volumetric flask and then raising the volume to the mark with mobile phase. For constructing calibration curve, a series of reference standards containing 20, 40, 60, 80, 100, 120, 140 Mg/mL were prepared from the stock solution using mobile phase as the diluent.
Preparation of solution for spectrophotometry studies. For spectrophotometric analysis, stock solution containing 5.0 mg/mL glimepiride was prepared by dissolving 0.125 g glimepiride in 25 mL methanol in a volumetric flask. Different aliquots of stock solution equivalent to 400-1600 Mg were transferred into a series of 10 ml volumetric flasks. 1 mL of 2,3,5-triphe-nyl-2H- tetrazolium chloride (0.1%), 40 mL of sodium hydroxide solution (0.1 M) were added in each flask and the mixture were heated in a water bath at 60 ± 2°C for 30 min. The flasks were cooled and the volume was made to the mark with methanol. The absorbance was measured at 413.5 nm against a reagent blank.
Preparation of sample solution of glimepiride for HPLC analysis. Twenty tablets were ground to fine powder. A quantity equivalent to 50 mg of glimepiride was placed in a 50 mL volumetric flask and the volume was made up to 50 mL with mobile phase. After filtration, the solution was diluted with mobile phase to get a final concentration of 80 Mg/mL.
Robustness. Robustness is a slight change in the conditions of analysis. Robustness of HPLC method was determined by variation of different parameters like change in mobile phase composition and pH value of the formic acid solution. In addition, the stability of the analytical solutions was also examined by analyzing them after 12 and 24 hrs. For spectrophotometric method, the robustness was evaluated by variation in the concentration of reagent and sodium hydroxide, temperature and heating time before measuring the absorbance.
Specificity. Specificity of the HPLC and spectropho-tometric method was performed by preparing a synthetic mixture containing 50 mg of glimepiride and 50 mg of each starch, Kollidon CL, magnesium stearate and avicel present in tablet formulation of the drug. All were accurately weighed, transferred to two 50 mL volumetric flasks and dissolved by shaking with mobile phase and methanol, respectively, for HPLC and spec-trophotometric studies. These solutions were filtered and further diluted as required.
Linearity. The linearity of the proposed methods was checked by analyzing seven solutions in the range of 20 to 140 Mg/mL for HPLC and 40 to 160 Mg/mL for spectrophotometric method. The solutions were prepared in triplicate and the linearity was evaluated from linear regression equation.
Precision. Glimepiride standard solutions were analyzed five times within same day (intra-day precision) to obtain the repeatability and three times over different days (inter-day precision) to obtain reproducibility for each method.
Accuracy. Accuracy of the methods was checked by adding known concentration of glimepiride to sample solution and then comparing the results of calculated and measured concentrations. Sample solution of glimepiri-de (1 mg/mL) was prepared by dissolving the drug in mobile phase for HPLC analysis and in methanol for derivative spectrophotometric method. From the filtered solutions, aliquots of 1 mL were transferred to 50 mL volumetric flasks, containing 1.0, 3.0 and 5.0 mL glime-piride standard solution (1 mg/mL) and then the volume was made up to the mark with the respective solvent to obtain the final concentrations of 40, 80, and 120 Mg/mL, which corresponds to 50, 100, and 150% of the nominal analytical concentration of 80 Mg/mL. Each of these concentrations was made in triplicate.
RESULTS AND DISCUSSION
Glimepiride is a 2nd generation sulphonylurea an-tidiabetic agent used for the treatment of non-insulin dependant diabetes mellitus (NIDDM). The majority of reported methods for the determination of glimepiride are complex and time consuming. In this paper two novel analytical methods based on HPLC, and spectro-photometry have been proposed to quantify glimepiride in tablet formulations. For HPLC, symmetrical sharp peaks were obtained for glimepiride at 7.08 min. For spectrophotometric method the first derivative of gliepiride with 2,3,5-triphenyl-2H-tetrazolium chloride showed absorption maximum at 413.5 nm (Figure). Both of these methods are simple, less time consuming and are validated according to robustness, linearity, precision, accuracy and specificity.
To study the robustness of proposed HPLC method, intentional modifications in composition and pH value of formic acid solution used were made and the results are presented in Table 1. It was observed that change of ±0.5 unit of pH value around 3.50 had no impact on chromatographic performance. When amount of aceto-nitrile was reduced to 70% the retention time was increased to 9.28 min, whereas increasing the acetonitrile content to 90% reduces the retention time to 5.68 min.
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