Work plan & Methodology: HPLC Method Development The HPLC analytical Method developed on the basis of it s chemical structure, Therapeutic category, Molecular weight formula, pka value of molecule, nature, sensitivity, storage condition, and wave length of maximum absorption etc. (19) 1. Solubility - selecting specific solubility of active pharmaceutical ingredient of finish product in to general solvent like water,methanol,actonitrile,chloroform,0.1m sodium hydroxide solution, 0.1 M Hydroxide solution, Phosphate buffer,acetate Buffer having different ph we decide to select appropriate diluents. (19) 2. Mobile Phase-Then it is required to determine Pka value for respective molecule to select proper buffer for Mobile phase. In mobile phase we use Different types of buffer, Solvents and different combination of these both. (19) 3. Column-Then selection of column is an important thing for the development of any HPLC method; there are different types of column available in markets like water symmetry, Hypersil BDS, Hypersil ODS, Inrtsil ODS, Xterra Rp18, Xterra RP8, Reproshels, and Luna of different length. The selection of column depend upon the category of molecule i.e. were the molecule is acidic, basic or neutral in nature. 4. After selection of above it require to select proper parameter of instrument like flow, column oven temperature, sample tray temperature, wave length, injection volume, run time and then trial carry out at different condition still main drug and impurities do not resolved. (19) 5. It is require to develop method such manner that the theoretical plates, resolution, tailing, peak purity and the recovery should be within limit as norms and condition (19) Method Validation - Any new develop method of analysis required to validate before its application.it should carried out to reproducibility of results on any system and at any condition. The HPLC Method Will be validated ac per ICH and pharmacopeial Guide line (20) The Validation Parameter is as follow:
a) Specificity: specificity is ability to measure quantitatively the analyte in presence of component that may be expected to be present in the sample matrix. It ensures that there is no interference from diluents or degradation product or impurities or placebo (inactive material) or the analyte with each other. Selectivity shall be performed by injecting blank solution, placebo solution and all the known impurities at limit concentration as a part of selectivity (specificity). (20) b) Limit of Detection(LOD)and limit of quantification: Limit of detection is the lowest concentration of analyte in sample that can be detected But not necessarily quantitated. Limit of quantification is the lowest concentration of Analyte in a sample that can be determine with acceptable precision and accurately under The stated experimental condition. c) Linearity and Range : Linearity is measure of method ability to obtain results which are eighter directly or after Mathematical transformation proportional to concentration of the analyte within given Range.the range for linearity study is generally selected on the type of Experiment. Linearity should be performed from 50 % to 150% of the test concentration. 1. In Assay test linearity graph shall be plotted peak area response against standard concentration (20) 2. In impurity determination Linearity graph of principle peak and known impurity shall be plotted for peak area response against standard or impurity concentration. 3. In dissolution linearity graph of principle peak shall be plotted for peak area response against analyte concentration. d) Accuracy : The accuracy of analytical method is the closeness of the results obtain by that method to the true value. The accuracy of analytical method should be established across its Range. e) Precision : The precision of analytical method is the degree of agreement among individual test
Results when the method is applied repeatedly to multiple sampling of a homogeneous sample. Precision is a parameter that measures the degree of repeatability of method from the sample population under the normal operating circumstance. Relative standard Deviation (RSD) in percent is generally used to represent the precision of the method for sample between two sets of experiments (different days /different analyst/different instrument or combination of any of them). f) Robustness: Robustness of a method is its ability to derive accurate results even when there is slight change in the condition such as temperature, ph, buffer concentration, wavelength etc. Examples of typical variations are: 1) ph of mobile phase / buffer. 2) Same make of column and different lot number or different columns makes. 3) Column oven temperature. 4) Flow rate. 5) Wavelength. 6) Mobile phase composition. 7) RPM of dissolution machine. 8) ph of dissolution medium. NOTE: For Chromatographic parameter variation Any three or all of the above stated variations ( from point 1 to 6 ) as appropriate may form a part of the robustness study. For Dissolution parameter variation Any one or all the above stated variations (from point 7 to 8) as appropriate may form a part of the robustness study. g) Forced Degradation : Forced degradation is an important aspect in development of stability indicating method. in this parameter the product or the sample should degrades forcefully as they show the degradation of main drug which is converted in to impurities. Degrade the sample such manner that the peak due to degradation of product should be well resolved from principle peak and it s
results in to the stability indicating method. The degradation should be finding in any one of the following. 1. Acid Degradation. 2. Alkali degradation 3. Peroxide Degradation 4. Humidity Degradation. 5. Thermal degradation. 6. Photolytic Degradation. h) Filter validation: Filter validation is to be carried out to evaluate the effect of filtration of the analyte on the analysis. Most of prepare solution are turbid so direct inject it in to instrument is harmful to instrument as well as column also. Due to unfiltered material it is possibility to damage of column and blocking of injector needle. In this parameter the absolute difference between filter and unfiltered results not more than 2 %. i) Solution Stability: This experiment provides the information about stability of analyte in the Sample and standard solution at room temperature if the material is unstable at room temperature the same can be assessed at refrigerated condition or as given in the method of analysis for respective product. III) Preparation of validation summary reports: After validation summary report will be prepared for all parameter of respective product.
Work plan:- First Year: literature study and collection of experimental material First six month - literature searching for availability or non availability of HPLC methods. Second six month - Collection of sample, placebo, impurities, Column, chemicals etc Second Year: Development and validation First six month - Development of analytical method s for respective test. Second six month - validation for developed method and reporting of its results.