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 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 short cut 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.
HPLC Method Development, Validation, and Analysis: From Drug Discovery to Clinical Trials
HPLC method development is required at each phase of drug R&D, starting from the early discovery phase. During this stage, the HPLC method can be developed utilizing a generic protocol as the drug is being analyzed at a higher concentration and only semi-quantitative values are needed. Naturally, this fit-for-purpose approach helps lower the HPLC test cost significantly during early discovery studies. As the drug advances in the next phase, however, it becomes crucial to optimize the method and make it more reproducible. HPLC method validation becomes necessary when Tox studies are planned for preparing IND application filing. These methods developed properly can be promptly validated. Therefore, we must prioritize robust HPLC method development and analysis even during the earlier stages. This way, the methods used in preclinical phase can be seamlessly transferred to the clinical programs and would not require extensive HPLC method development and validation.
