ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ALOGLIPTIN AND METFORMIN HYDROCHLORIDE TABLET DOSAGE FORM BY RP-HPLC METHOD
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1 Indexed in Cite Factor - Directory of International Research Journals in association with leading universities ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ALOGLIPTIN AND METFORMIN HYDROCHLORIDE TABLET DOSAGE FORM BY RP-HPLC METHOD A. PRAVEEN KUMAR* 1, G. ARUNA 2, K. RAJASEKAR 3, P. JAYACHANDRA REDDY 4 ABSTRACT In the present work, RP-HPLC method for two drugs have been developed and validated for simultaneous determination of Alogliptin and Metformin Hydrochloride in Tablet dosage form. In this method, 0.2% TEA ph adjusted with OPA to 6.0 was used as buffer mobile phase was prepared by adjusting the ratio of buffer and Methanol as 30:70 v/v + 0.2% Triethylamine and a flow rate of 1.0 ml/min. The optimum wavelength for detection was 254 nm and a run time of 10 minutes was used. The column used was Agilent C 18 with dimension 250 mm length, 4.6 mm i.d., 5µ particle size. The method was validated for its linearity, accuracy, precision, specificity, robustness. The system suitability parameters passed and linearity was observed in the range of µg/ml for Alogliptin and Metformin Hydrochloride respectively and the correlation coefficient of Alogliptin and Metformin Hydrochloride were and The accuracy was performed and the % recovery was observed as 99.96% and 99.83% for Alogliptin and Metformin Hydrochloride respectively. Thus a novel, sensitive, accurate, specific, precise method was developed for the Simultaneous determination of Alogliptin and Metformin Hydrochloride in tablet dosage form by RP-HPLC Method. KEYWORDS Alogliptin, Metformin, Reverse Phase HPLC, Accuracy, Linearity, Tablet Dosage Form. AFFILIATION Krishna Teja Pharmacy College, Department of Pharmaceutical Analysis, Tirupati, Andhra Pradesh, India. *Author for Correspondence ID: Praveenorkumar6@gmail.com; Mob:
2 INTRODUCTION Alogliptin is an orally administered anti-diabetic drug in the DPP-4 inhibitor class. Molecular Formula C 18 H 21 N 5 O 2 and IUPAC name 2-({6-[(3R)-3-aminopiperidin-1-yl]-3-methyl-2,4- dioxo-3,4- dihydropyrimidin-1(2h)- yl}methyl)benzonitrile. Metformin Hydrochloride is an oral antidiabetic drug in the biguanide class. It is the first-line drug of choice for the treatment of type 2 diabetes, in particular, in overweight and obese people and those with normal kidney function Its use in gestational diabetes has been limited by safety concerns. It is also used in the treatment of polycystic ovary syndrome, and has been investigated for other diseases where insulin resistance may be an important factor. Metformin Hydrochloride works by suppressing glucose production by the liver. Molecular Formula C 4 H 11 N 5.HCl and IUPAC name 1-carbamimidamido- N, N-dimethyl methanimidamide The review of literature revealed that various analytical methods involving spectrophotometry, HPLC, have been reported for Metformin in single form and in combination with other drugs. Several analytical methods have been reported for Alogliptin in spectrophotometry, HPLC. To date, there have been no published reports about the simultaneous estimation of Alogliptin and Metformin Hydrochloride by HPLC in bulk drug and in pharmaceutical dosage forms. This present study reports for the first time simultaneous estimation of Alogliptin and Metformin Hydrochloride by HPLC in bulk drug and in pharmaceutical dosage form. The proposed method is validated as per ICH guidelines. Fig. 1: Structure of Alogliptin. Fig. 2: Structure of Metformin Hydrochloride. 59
3 MATERIALS AND METHODS Instrumentation HPLC system (Agilent HPLC Model-1220 with Chemstation Software) containing C 18 (Agilent 250 x 4.6 mm, 5µ) column with UV- VWD detection. Analytical UV-1600 Plus double beam spectrophotometer with a fixed slit width 1nm and 1cm matched quartz cells was used for all the spectral measurements. Chemicals and reagents Analytically pure Alogliptin was kindly provided by Active pharma Pvt. Ltd, Hyderabad and Metformin Hydrochloride was provided by Kaushik Therapeutics Pvt.Ltd, Hyderabad as gift samples. Analytical grade methanol was purchased from Merck & Co. Glasswares used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in hot air oven. Water (HPLC grade) were purchased from Merck, India. Triple distilled water is used for all purpose. Chromatographic conditions The mobile phase consisted of methanol and water in ratio 70:30, 0.2% TEA is added to water and ph was adjusted to 6.0 with OPA. The contents of the mobile phase were filtered before use through a 0.45μ membrane and degassed for 10 min. The mobile phase was pumped from the solvent reservoir to the column at a flow rate of 1.0 ml/min and the injection volume was 10 μl. The column temperature was maintained at ambient temperature. The eluents were monitored at 254 nm. Preparation of standard stock solutions Accurately weighed 10 mg of Alogliptin and Metformin Hydrochloride standard were transferred to separate 10 ml volumetric flask and dissolved in 10 ml methanol. The flasks were shaken and volume was made up to the mark with methanol to give solutions containing 1000 μg/ml Alogliptin and 1000 μg/ml Metformin Hydrochloride. From this solution 0.5ml was transferred to volumetric flask of 10 ml capacity. Volume was made up to the mark to give a solution containing 50μg/ml of Alogliptin and 50μg/ml Metformin Hydrochloride. Calibration of standards The standard calibration curve was constructed for Alogliptin and Metformin Hydrochloride Different volumes of stock solutions of each were accurately transferred in to 10mL volumetric flasks and diluted to mark to yield a concentration range of μg/ml solutions of Alogliptin and μg/ml solutions of Metformin Hydrochloride. The calibration line was obtained by plotting the peak area against concentration of drug. The results were shown in table 3. Method validation The proposed method has been extensively validated in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility. The accuracy was expressed in terms of percent recovery of the known 60
4 amount of the standard drugs added to the known amount of the pharmaceutical dosage forms. The precision (Coefficient of Variation - C.V.) was expressed with respect to the repeatability, intra-day and inter-day variation in the expected drug concentrations. After validation, the developed methods have been applied to pharmaceutical dosage form. System Suitability Criteria It is defined as tests to measure the method that can generate result of acceptable accuracy and precision. The system suitability was carried out after the method development and validation have been completed. The system suitability was assessed by five replicate analyses of the drugs at concentrations of 50 μg ml 1 of Alogliptin and 50 μg ml 1 of Metformin Hydrochloride and for this, parameters like plate number (n), tailing factor, peak asymmetry of samples were measured, and shown in table 3. Method Validation parameters Method was validated as per ICH (Q2) guidelines with respect to linearity, accuracy, precision, specificity, robustness, limit of detection and limit of quantification. a) Specificity Commonly used excipients (starch, microcrystalline cellulose and magnesium stearate) were spiked into a pre weighed quantity of drugs. Specificity of the method was shown by quantifying the analyte of interest in the presence of matrix and other components. Blank injections have shown no peaks at retention time of 3.30 min and 6.26 min, the proposed method was specific for the detection of Alogliptin and Metformin Hydrochloride respectively. b) Linearity Appropriate volume of aliquot from Alogliptin and Metformin Hydrochloride standard stock solution was transferred to volumetric flask of 10 ml capacity. The volume was adjusted to the mark with methanol to give a solutions containing μg/ml Alogliptin and μg/ml Metformin Hydrochloride. The slope, Y-intercept and correlation coefficient were calculated. The regression line relating standard concentrations of drug using regression analysis, the calibration curves were linear in the studied range and equations of the regression analysis were obtained: y = x ; R² = ; Alogliptin and y = 10.46x ; R² = ; for Metformin Hydrochloride respectively. The mean and correlation coefficient of standard curves (N=3) were calculated. The represented data was shown in below figure 4, 5 and Table 1 & 1A Table 1: Linearity data of Alogliptin. Concentration (µg/ml) Peak Area Statistical Analysis Slope: Intercept: Correlation coefficient:
5 Table 1A: Linearity data of Metformin Hydrochloride. Concentration (µg/ml) Peak Area Statistical Analysis Slope: Intercept: Correlation coefficient: c) Accuracy Accuracy was assessed by determination of the recovery of the method by addition of standard drug to the pre-quantified placebo preparation at 3 different concentration levels 80, 100 and 120 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed 3 times and average recoveries were measured. Results of assay and recovery were presented in the Table 2. Table 2: Accuracy Data of Alogliptin and Metformin Hydrochloride. Drug % Recovery Conc of sample(µg) Avg area of sample Amount recovery % Recovery Mean % Recovery Metformin Hydrochloride 80% % % Alogliptin 80% % % d) Precision The repeatability was evaluated by assaying 6 times of sample solution prepared for assay determination. The intraday and interday precision study of Alogliptin and Metformin Hydrochloride was carried out by estimating different concentrations of Alogliptin (50 62
6 μg/ml) and Metformin Hydrochloride (50 μg/ml), 3 times on the same day and on 3 different days (first, second, third) and the results are reported. The results are shown in Table 3. Table 3: Precision studies of Alogliptin and Metformin Hydrochloride. Parameter Metformin Hydrochloride Alogliptin System precision (%RSD) Method precision(%rsd) Intermediate precision(%rsd) e) Robustness The robustness of the method was evaluated by analyzing the system suitability standards and evaluating system suitability parameter data after varying the HPLC pump flow rate (±0.2ml) and organic solvent content (±2ml) and ph (±0.2). None of the alterations caused a significant change in peak area R.S.D (%), USP tailing factor and theoretical plates. Although the changes in retention times were more significant, and quantification was still possible. Results of robustness studies are shown in Table 4 & 5. Table 4: Peak Results for Robustness for Alogliptin. S.No. Parameter Condition % RSD System suitability results USP Symmetry USP Plate Count Flow rate by ± 2% ph of Buffer solution by ± 0.2 units Wavelength of analysis ± 2nm
7 Table 5: Peak Results for Robustness for Metformin Hydrochloride. S.No. Parameter Condition % RSD System suitability results USP Symmetry USP Plate Count 0.8 ml Flow rate by ± 2% 1.0 ml ph of Buffer solution by ± 0.2 units Wavelength of analysis ± 2nm 1.2 ml nm nm nm f) LOD and LOQ LOD and LOQ were calculated from the formula 3.3 x (σ/s) and 10 x (σ/s), respectively where, σ is standard deviation of intercept and S is the mean of slope. The LOD and LOQ can also be determined by S/N. The value for LOD should be 3-5 whilst for LOQ R ESULTS Optimization of mobile phase was performed based on resolution of the drugs, asymmetric factor and theoretical plates obtained for Alogliptin and Metformin Hydrochloride (Figure 3). Figure 3 Optimized chromatogram of Alogliptin and Metformin Hydrochloride 64
8 The mobile phase consisted of methanol and water in ratio 70:30 (0.2%TEA is added to water and ph was adjusted to 6.0 with OPA) was selected which gave sharp, well-resolved peaks for Alogliptin and Metformin Hydrochloride. The retention times for Alogliptin and Metformin Hydrochloride were 3.30 and 6.26 min respectively. UV overline spectra of Alogliptin and Metformin Hydrochloride showed that the both the drugs absorbed appreciably at 254 nm, so the same was selected as the detection wave length during the studies. The calibration curves was found to be linear over the range of μg/ml for Alogliptin and μg/ml for Metformin Hydrochloride. The data of regression analysis of the calibration curves are shown in figure 4 and 5. Fig. 4: Linearity plot of Alogliptin. 65
9 Figure 5 Linearity plot of Metformin Hydrochloride Intra-day precision studies were carried out. For intra-day studies RSD (%) values were found to be for Alogliptin and for Metformin Hydrochloride respectively. For inter-day studies RSD (%) values were found to be for Alogliptin and for Metformin Hydrochloride respectively, the low RSD (%) value indicate that the method is precise. The detection limits for Alogliptin and Metformin Hydrochloride were μg. ml -1 and μg. ml -1 respectively, while quantitation limits for Alogliptin and Metformin Hydrochloride were μg. ml -1 and μg. ml -1 respectively. The above data shows that a microgram quantity of both the drugs can be accurately and precisely determined. The validation parameters are summarized in table and the system suitability test parameters are shown in table 3. Robustness of the method was studied by changing the flow rate of the mobile phase from 1 ml. min -1 to 0.8 ml. min -1 and 1.2 ml. min -1. And by changing the Wavelength 252nm and 256nm was studied. The ph of moble phase also changed to 5.8, 6.2 and the results were studied CONCLUSION The present study represents an accurate, precise and specific HPLC method for routine analysis of Alogliptin and Metformin Hydrochloride tablet dosage form. In addition to assay it may be used to detect related substance or other impurities which are formed during storage conditions and the analyte of interest could be estimated without any interferences. The use of C 18 column in the present work has shown better elution of analytes with good resolution, improved plate count, capacity factor, reduced tailing. So the C 18 column can be used to achieve high specificity in shorter time of analysis of Alogliptin and Metformin Hydrochloride in injectables as per ICH Q2 (R2) guidelines. ACKNOWLEDGEMENT The authors would like to thank P. Jayachandra Reddy, M.Pharm., Ph.D. FIC. Principal, Krishna Teja Pharmacy College for providing necessary facilities to carry out the work. 66
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