Bio-Analytical Assay Methods for Estimation of Drugs in Human Plasma using LC–MS/MS: A Review

Bio-analytical assay methods play a crucial role in the quantitative estimation of drugs in biological matrices for pharmacokinetic, bioequivalence, and therapeutic monitoring studies. Among various analytical techniques, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has emerged as a highly sensitive, selective, and robust tool for drug analysis in human plasma. This review provides a comprehensive overview of bio-analytical methods employed for the estimation of drugs in biological matrices using LC–MS/MS.The review discusses the two fundamental components of bio-analytical method development, namely sample preparation and instrumental analysis. Various extraction techniques, including protein precipitation, liquid–liquid extraction, and solid-phase extraction, are critically evaluated with respect to their efficiency in sample clean-up and analyte recovery. The principles of chromatographic separation and mass spectrometric detection, including ionization modes and multiple reaction monitoring, are also summarized. Furthermore, this article highlights regulatory requirements for bio-analytical method validation as outlined by international agencies such as the US Food and Drug Administration (USFDA) and European Medicines Agency (EMA). Key validation parameters including selectivity, sensitivity, linearity, accuracy, precision, recovery, and stability are discussed in detail. The application of validated LC–MS/MS methods in the quantification of drugs in human plasma for clinical and bioequivalence studies is also reviewed. Overall, this review emphasizes the significance of LC–MS/MS–based bio-analytical methods in modern drug development and clinical research.