International Journal of Innovative Pharmaceutical Sciences and Research
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1 International Journal of Innovative Pharmaceutical Sciences and Research METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTITATION ESTIMATION OF DILTIAZEM HCL IN TABLET DOSAGE FORM BY RP-UPLC I Deepa Bajetha*, 1 Bharti Mangla, 2 Suresh kumar joshi 1 Advanced Institute of Pharmacy, Palwal (Haryana), INDIA 2 B.S Anangpuria Institute of Pharmacy, Faridabad (Haryana), INDIA Abstract A reverse phase Ultra Performance Liquid Chromatographic method was developed and validated for the quantitation of an antihypertensive drug diltiazem HCl in bulk and pharmaceutical dosage form. The analysis was performed on Waters ACQUITY BEH C18 column (50 x 2.1 mm, 1.8μm) with mobile phase composed of ammonium acetate (5 mm, neutral) and acetonitrile in gradient mode at a flow rate of 0.6 ml/min with detection of analyte at 210 nm. The separation was achieved within ± min. The method validation parameters showed good results for linearity, accuracy, and precision. The calibration curve was linear in the concentration range μg/ml with coefficient of correlation The mean recovery was found to be %. The proposed method was validated as per ICH guidelines for various parameters like accuracy, precision, linearity and robustness. Keywords: Ultra Performance liquid chromatography, Diltiazem, calcium channel blocker, Precision, Accuracy, Robustness Corresponding Author: Suresh kumar joshi B.S Anangpuria institute of pharmacy, Faridabad (Haryana), India, INDIA suresh92joshi@gmail.com Phone: , Available online: April Issue 340
2 INTRODUCTION Diltiazem HCl is a non-dihydropyridine (non-dhp) calcium channel blockers, used in the treatment of hypertension, angina pectoris, and also used in some types of arrhythmia. It is a benzothiazepine, which differentiates it from the additional classes of non-dhp calcium channel blockers for instance the phenylalkylamines [1-3] Diltiazem is an important coronary vasodilator drug belongs to calcium channel blocker. Chemically, diltiazem HCl is, (+)-cis-1,5-benzothiazepin-4-(5h)one,3-(acetyloxy)-5-[2- (dimethylamino)ethyl]-2,3-dihydro-2-(4-methoxy phenyl)-monohydrochloride treatment of hypertension. [2] HPTLC [3] method is reported alone and in combination with other drugs. So far no method has been reported for spectroscopic estimation of Diltiazem HCl; present work is an attempt to develop simple, precise, economic, reproducible method for estimation of Diltiazem hydrochloride [4-6]. MATERIALS AND METHODS Reagents and Chemicals: Milli Q water, HPLC grade Methanol, Acetonitrile and Ammonium Acetate was used in the study. Chromatographic Condition: An Agilent Technology 1290 Infinity UHPLC equipped with Photodiode Array detector, the purity determination performed on a stainless steel column 50mm long, 2.1 mm internal diameter filled with Octadecylsilane (ODS) which was chemically bonded to porous silica particles of 1.8μm diameter C18 column (Acquity C18, 2.1x50mm,1.8μm particle size) [35,36]. The mobile phase consisting of mg Ammonium Acetate in 1000 ml Mill-Q water and Acetonitrile in a 98:2 ratio & filtered by 0.45μ 6,6 Nylon Membrane filter paper and degassed [7-10]. Preparation of Blank Solution of DZM: Mixed well Methanol: Acetonitrile in a ratio of (50:50) sonicated and degassed. Preparation of Standard Solution of DZM: Weighed accurately and transferred about mg of Diltiazem HCl standard in a ml volumetric flask. Added a little quantity of primary diluent to dissolve, sonicated and degassed. Cooled and diluted to volume with secondary diluent [11-12]. Available online: April Issue 341
3 Preparation of Sample Solution of DZM: 20 tablets of Diltiazem HCl Hydrochloride each containing 60 mg of Diltiazem HCl were weighed and powdered. To prepare 200μg/ml concentration of sample solution, a quantity of powder equivalent to 20 mg (62.1 mg) was weighed approximately and transferred in a 50 ml of dried volumetric flask. The sample was initially dissolved in primary diluent and sonicated for 15 min. The volume was made up to 50ml and filtered by 0.2μm Nylon filters. Then approx. 5ml ml of this solution was further diluted to ml with secondary diluent to get the final concentration of 200μg/ml. 1 μl of this was injected into the column and chromatogram was recorded [12, 13]. Method development: To develop a sensitive, selective and simple assay method for the quantification and extraction of diltiazem during method development, different options were evaluated and to optimize for detection to chromatography parameters. First we have to find out the wavelength using the references. Then select the mobile phase varying the composition and ph, optimize it. Further optimization in chromatography conditions results to increase the response of analyte. A mobile phase i.e. 10 mm ammonium acetate buffer when combined with acetonitrile resulted in improved response. [11] Validation Parameter: The developed method had been validated in terms of different parameters such as: [12-15] Parameter Diltiazem HCl Accuracy: Recovery Studies Precision(RSD, n=5) Linearity and range(μg/ml) 3-30 Limit of detection(μg/ml) 7.96 Limit of Quantitation(μg/ml) Intraday Precision(RSD, n=3) Interday Precision(RSD, n=3) Selectivity: It is the analytical method to differentiate and quantify of the analyte in the presence of other components in the samples. For the selectivity, analysis of blank samples of the suitable Available online: April Issue 342
4 biological matrix should be obtained nevertheless from six sources. Each blank sample should be tested for selectivity and interference should be ensured at the lower limit of quantification. [6] Specificity: The ICH documents defines the specificity that the ability to assess clearly the analyte in the presence of components that may be probable to be present, such as the degradation products, impurities and matrix components. Linearity & Range: The linearity of an analytical method be present its ability to bring about the test results that are directly or by a well-defined mathematical transformation, proportional to the concentration of analyte present in the samples within a given range [15-17]. A linear relationship should be evaluated through the range of the analytical procedure. It may be demonstrated as the dilution of standard stock solution of active substances and synthetic mixtures of the product components by using the proposed procedure. The linearity should be evaluated by visual examination of a plot of signals as a function of analyte concentration. The slope, correlation coefficient and y-intercept of the regression line and residual sum of squares have to be submitted. For the setting up of linearity, a minimum of 5 concentrations is recommended. Acceptance criteria- the calculated value of r should pass the test for correlation. [18] Accuracy: The accuracy of an analytical method is the closeness of test results achieved by that method to the true value and the accuracy of the method was established by studying the recovery. The accuracy of the analytical method should be developed across its range. Accuracy is determined by replicate analysis of samples containing known concentration or amounts of the analyte. It should be measured using a minimum of five determinations per concentration. A least possible of three concentrations lay in the range of expected concentrations is recommended. [18-19] Precision: The precision of an analytical method is defined as the degree of agreement among individual test results when the method is applied repeatedly for multiple samplings of a uniform sample. It is generally expressed as the coefficient of variation (relative standard deviation) of a sequence of measurements. The precision may be measured of either the degree of both repeatability or reproducibility of the analytical method under normal operational conditions. [20] Available online: April Issue 343
5 Robustness: Robustness is defined as masseur of the ability of analytical method an analytical procedure to remain unpretentious by small but deliberate variation in method parameter (ph, mobile phase composition, temperature and instrumental setting) and provides an indication of its reliability during normal usages. [18] System Suitability Parameter: System suitability parameter is the evaluation of a composition of analytical system to show the performance to come across the standard required by the method. These parameter can be calculated experimentally to provide a quantities system suitability test report quantity of theoretical plates (efficacy ), resolution, capacity factor, separation (relative retention), telling factor relative standard deviatation (precision). [10] RESULTS Specificity: The specificity of method was determined by comparing the chromatograms obtained from the sample containing Diltiazem HCl standard stock with those obtained from test sample of Diltiazem HCl and blank of that. The specificity study shown at the absence of interference of impurities with the drug since no extra peak appeared at the RT of drug. Figure 1: Representative chromatogram of Standard Solution Figure 2: Representative chromatogram of Sample Solution Available online: April Issue 344
6 Linearity: Linearity was determined by injecting six replicate injections of standard solutions of Diltiazem HCl (200 ppm) to check the system suitability. The response was measured as peak area. The calibration plot was generated by replicate analysis at six concentration levels and the linear regression equation was calculated by using the least square method in Microsoft Excel program. Figure 2: linearity Plot Different day precision: Table 1: Results of precision study of Diltiazem Hydrochloride Results of accuracy study: The accuracy of method was determined by calculation of % recovery. Recovery is typically determined by comparing the response of the method with a reference material with the known value assign to the material. If the recovery of the assay was poor (e.g. less than 90%) it would be a good indication that there is a problem with the method. The developed method for Diltiazem HCl is a valid method. Available online: April Issue 345
7 Table 2: Results of accuracy study Solution stability study: A drug solution of 200 ppm was prepared and kept at room temperature i.e. 25 o C for 24 hrs. After that drug solution was analyzed and it was found to be stable at room temperature. Table 3: Solution stability study Robustness: It is the capacity of a method to remain unaffected by small deliberate variations in method parameters. Such as buffer content (±2mM), temperature ( 2 C). The results are presented in Table 4 Table 5 along with system suitability parameters of normal methodology. Table 4: Calculation for Increasing the Temperature of the Auto sampler by 2 C Available online: April Issue 346
8 Table 5: Calculation for Decreasing the Temperature of the Auto sampler by 2 C Table 6: Calculation for Increasing the Buffer Concentration from 5mM to 7mM Table 7: Calculation for Decreasing the Buffer Concentration from 5mM to 3mM Available online: April Issue 347
9 SUMMARY: Table 8: Validation report summary S.No. Parameter Experiment Discussion Acceptance criteria 1 Specificity Blank interference System suitability 1) No interference was observed at retention time of Diltiazem HCl 2) The relative standard deviation for six replicate measurements of peak area response of standard preparation was found to be ) The relative standard deviation for retention time of six replicate injections of standard preparation was found to be ) No interference should be observed at retention time of Diltiazem HCl. 2) The relative standard deviation for six replicate measurements of peak area response of standard preparation should be not more than 2.0%. 3) The relative standard deviation for retention time of six replicate injections of peak should not be more than 1.0%. 2 Linearity 1) Coefficient of correlation (r) 2) Y intercept ratio ± Precision 1) Precision of the system 1) The relative standard deviation for six replicate measurements of peak area response of standard preparation was found to be ) The relative standard deviation for retention time of six replicate injections of standard preparation was found to be ) The relative standard deviation for six replicate measurements of peak area response of standard preparation should be not more than 2.0%. 2) The relative standard deviation for retention time of six replicate injections of peak should not be more than 1.0%. 2) Precision of six replicate samples prepared. 1) The relative standard deviation for six replicate preparations was found to be ) The relative standard deviation for six replicate preparations should be not more than 2.0%. Available online: April Issue 348
10 4 Reproduc-ibility (Intermed-iate Precision) Analyst to analyst The %RSD for % assay obtained from 6 precision samples was found to be The %RSD for % assay obtained from 6 precision samples should be not more than 2.0%. 5 Accuracy Recovery study 6 Solution stability The individual recoveries were found to be between to %. 1) The %Cumulative RSD for standard area and % assay of sample at initial hr and at predetermined time intervals at room temperature was found to be )The pattern of chromatography remained same throughout solution stability study. All the individual recoveries should be within 95.0% to 105.0% 1) The Cumulative RSD for standard area and % assay of sample at initial hr and at predetermined time intervals should be not more than 2.0% 2)The pattern of chromatography should remain same throughout solution stability study. 7 Robustness 1) Change in autosampler temperature from 40 2 C 2) Change in Buffers conc. from 5mM ± 2 mm The Cumulative RSD for % assay obtained from precision and robustness study was found 1.9. The Cumulative RSD for % assay obtained from precision and robustness study was found to be The Cumulative RSD for % assay obtained from precision and robustness study should be not more than 2.0%. The Cumulative RSD for % assay obtained from precision and robustness study should be not more than 2.0%. Standard drug sample of Diltiazem HCl was identified and characterized by various spectroscopic methods and it was found to be highly pure. Standard sample of drug was utilized as reference standard. Parameters such as detection wavelength, different combinations of different organic solvent and buffers at various ph, composition and flow rate were studied. Feasibility of various mobile phase compositions with various concentrations and ph range along with altered flow rate was tested for complete resolution of component. Available online: April Issue 349
11 Rapidity of method was further checked with two columns viz. Phenomenex luna R C18 (2.1 50) mm, 1.8µm and ACQUITY BEH C18 (2.1 50) mm, 1.8µm. It was found that chromatographic resolution, selectivity, sensitivity and rapidity were good with ACQUITY BEH C18 (2.1 50) mm, 1.8µm column. For quantification purpose dilution of Diltiazem HCl in linear range. Optimized mobile phase was Acetonitrile: 5mM Ammonium Acetate buffer (gradient mode), neutral. Reversed phase C18, ACQUITY BEH C18 (2.1 50) mm, 1.8µm was selected for further analysis. Flow rate was 0.60mL/min throughout the run time. Quantification was done by UV detector at 210nm. Developed method is reversed phase UPLC method in which column stationary phase is non-polar and polar mobile phase, so eluted components from the column is Diltiazem HCl (R t 1.06 min). Validation of the develop and optimized UPLC method was carried out in the light of ICH Guidelines with respect to parameters- linearity, specificity, selectivity and inter and intra-day variability (precision and accuracy). The linear regression equation for Diltiazem HCl was found to be y = x and the regression coefficient values was found to be indicating a high degree of linearity. The proposed method was stable during analysis. The stability of the analyte solution was determined at interval of 0hr, 2hr, 4hr, 8 hr, 12hr, and 24hr. The individual recoveries were found to be between to % which showed that estimation of dosage forms were within the acceptances level of 95% to 105%. CONCLUSION: Developed assay method is simple, rapid, accurate, precise, economical, specific and reproducible for the quantitative determination of Diltiazem HCl with good resolution in short time and high sensitivity. The flow rate programming of isocratic mobile phase which censored on overall time of sample analysis and thereby made the method is more rapid and cost effective. The detection of analyte at their maximal absorbance wavelength made the method more sensitive. FUTURE SCOPE: Available online: April Issue 350
12 The established analytical method can be used for routine analysis of Diltiazem HCl in bulk dosage forms. REFERENCES 1. Shrivastava R. Spectrophotometric determination of diltiazem in pharmaceutical and invivo samples with Pd (II). Int. J Of Pharmaceutical Sci. And Research. 2013; 4(12): Dhavse VV. and Devrajan PV. High Performance Liquid Chromatographic method for Diltiazem Hydrochloride as bulk and in pharmaceutical preparations. J of Chromatography B: Biomed. Sci. and Application. 1998; 706(2): Baviskar D and Sharma R. High- Performance liquid chromatography method for determination of diltiazem in human plasma. Asian Journal of Chemistry. 2009; 21, (7): Vidya V. G and Chandewar. Develpoment and validation of spectroscopic method for estimation of diltiazem HCl in solid dosage form. Journal of Pharmacy Research, 2010; 3(12): Sudha T, Kanth K V, Nukala P et al. Method Development and Validation- A Review Journal of Advanced Pharmacy Education & Research. 2012; 2(3): Ahuja S, Dong M.W. Handbook of Pharmaceutical Analysis by HPLC, Published by Elsevier Academic Press. 2009; 6: The International Pharmacopoeia, Vol. 1, 2005, pp Anjaneyulu Y, Chandrashekhar K, Manickar V. A Textbook of Analytical Chemistry. 1 st Edition, Pharma Book Syndicate, Hyderabad. 2006: Sabir MA, moloy M and Singh, B P. Development and validation of HPLC method - a review. International Research of Pharmacy. 2013; 4(4). 10. Brambilla G, Ganderton D, Garzia R et al. Modulation of Aerosol Cloud Produced by Pressurized Inhalation Aerosols. International Journal of Pharmaceutics. 1999; 186: Cumar R. P. and Vasudevan M. HPLC method development and validation for the estimation of diltiazem in bulk and tablet dosage forms. Asian Journal of Pharmaceutical and Clinical Research. 2012; 5(3): 62. Available online: April Issue 351
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