The development, validation, and analysis of the HPLC or the High-Performance Liquid Chromatography are one of the most crucial techniques employed in the drug testing procedure of both the biological fluids and formulations. Usually, the first step of the HPLC method development process is reviewing whatever existing information you have about the given pharmaceutical ingredient under analysis. Thereafter, you need to move ahead with the selection of an appropriate HPLC column which helps the analyst in obtaining a sharp peak within a short interval of time. In this step itself, with the help of an appropriate column, a neat analyte solution is chromatographed. As the number of HPLC columns available are more beyond one’s count, the selection of your perfect pick is extremely difficult.
Process of the HPLC analytical Method Development –
HPLC analysis is accomplished through the following stages –
Literature Collection – Any analytical method development starts with the collection of sufficient back-up literature. Here, various pharmacopoeias are screened for detecting the availability of various analytical methods. The shortlisted methods are then developed and validated accordingly to find its compatibility to be employed for its intended use.
Sample Information – Both, the knowledge about nature and the sample type is critical for developing a new HPLC method. It is imperative for the analyst to know whether his sample is polar or non-polar.
Solvent selection – Next essential parameter of solvent selection is exploring whether your given compound is soluble or insoluble in water. The solvent should be selected in accordance with its solubility, and impurities and the degradation products present within your sample.
Selection of the stationary phase – Your stationary phase should be selected according to your column packing or bonded phase, which is either dependent on the molecular polarity or its by-products.
Selection of your mobile phase – This is one of the key parameters as it is known to encourage the interactions that take place between your solute and stationary phases.
Selection of detector – Your detector selection depends on the chromophores that are present or absent within your analyte sample.
If you aim at achieving a fast and effective drug analysis, it is quite imperative for you to plan your method development according to it. The general approach for any analytical method is easily available in the literature reference for the same including information on drug analysis, nature of the sample, solvent selection, selection and mobilization of mobile and stationary phases, and the type of detectors employed in the entire process. Additionally, modifying your mobile phases by addition of either the buffers or organic additives are important for improving the chromatographic separation, resolution, selectivity, and sensitivity of the test method employed. HPLC method development requires a thorough knowledge about both the solubility and pKa of the compound under analysis. This helps in the process of selecting the mobile phase and type of column packing and their subsequent optimization. Besides, HPLC method development also includes detection of the analytes, which is thereby selected by obtaining relevant information about the chemical nature of your analyte molecules.