What Is HPLC Method Development, Validation, and Analysis?
Foolproof HPLC Method Development, Validation, and Analysis for Reliable Peaks and Bioanalytical Data
High Performance Liquid Chromatography (HPLC) method development, validation, and analysis is one of the most widely used techniques for drug testing in formulations and biological fluids. Usually, the first step in HPLC method development is to review existing information given substantial research has already been published on this topic. Next, it is imperative to select a proper HPLC column that can provide a sharp peak with a reasonable retention time. In this step, a neat solution of the analyte is chromatographed using an appropriate column. There are countless choices of HPLC columns from different vendors, and the selection of an adequate column can itself be challenging. Afterward, we should select a suitable mobile phase for sound separation of analyte from impurities or endogenous material. Closer to the end, we deliberate over the detection technique for HPLC method development. This step, however, has become somewhat routine given the mass spectrometer has emerged as a popular choice for HPLC analysis due to its highly selective nature. Alternative detection modes require revisiting method development and further troubleshooting. Finally, careful sample preparation is also essential and must be reviewed thoroughly during HPLC method development. Often analysts take a shortcut for HPLC sample preparation and rely on the mass spectrometer for selective analyte detection. This practice, however, sometimes does not work out successfully due to ion suppression and a variety of other reasons. In summary, High-performance liquid chromatography (HPLC) Analysis is an analytical chemistry technique to separate, detect, and quantitate different drugs and metabolites within various formulations, vehicles, and biological matrices. For assay reproducibility, HPLC Method Development relies on the right combination of pumps, analytical columns and pre-columns, mobile phase, and autosampler conditions. HPLC Method Validation ensures proper assay selectivity, sensitivity, and reproducibility.