Recent Advances in Bioanalytical Methods for Quantitative Determination of Drugs in Biological Matrices: A Comprehensive Review

Abstract

Quantitative determination of drugs in biological fluids is fundamental to pharmaceutical research, clinical pharmacology, therapeutic drug monitoring, pharmacokinetic investigations, and regulatory submissions. Accurate measurement of drug concentrations in biological matrices such as plasma, serum, whole blood, urine, saliva, cerebrospinal fluid, and tissues is essential for evaluating drug absorption, distribution, metabolism, and excretion, assessing bioavailability and bioequivalence, optimizing dosage regimens, and ensuring drug safety and therapeutic efficacy. Over the past two decades, bioanalytical science has undergone remarkable advancements, transitioning from conventional analytical methods to highly sensitive and selective chromatographic and hyphenated techniques. Among these, high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UHPLC), gas chromatography–mass spectrometry (GC–MS), liquid chromatography–tandem mass spectrometry (LC–MS/MS), high-resolution mass spectrometry (HRMS), and capillary electrophoresis–mass spectrometry (CE–MS) have become indispensable tools for quantitative drug analysis. Efficient sample preparation remains a critical component of bioanalysis, with techniques such as protein precipitation, liquid–liquid extraction, solid-phase extraction, microextraction methods, and automated sample preparation significantly reducing matrix effects while improving analyte recovery, sensitivity, and reproducibility. Recent innovations, including microfluidic platforms, dried blood spot sampling, biosensors, nanotechnology-based analytical systems, high-resolution mass spectrometry, artificial intelligence-assisted data processing, and automated bioanalytical workflows, have further enhanced analytical performance, throughput, and reliability. In parallel, stringent bioanalytical method validation guidelines issued by regulatory authorities, including the International Council for Harmonisation (ICH M10), the United States Food and Drug Administration (US FDA), and the European Medicines Agency (EMA), have established standardized criteria for ensuring the accuracy, precision, selectivity, sensitivity, robustness, and reproducibility of bioanalytical methods. This comprehensive review critically discusses recent advances in bioanalytical techniques for the quantitative determination of drugs in biological fluids, emphasizing analytical principles, sample preparation strategies, hyphenated analytical technologies, bioanalytical method validation, regulatory expectations, and applications in pharmacokinetic, bioavailability, bioequivalence, toxicokinetic, and clinical studies. Furthermore, the review highlights current challenges, emerging trends, and future perspectives that are expected to shape the next generation of bioanalytical methodologies for pharmaceutical research and clinical applications.

Keywords: Advanced bioanalytical techniques; Drug quantification; Biological fluids; LC–MS/MS; UHPLC–MS/MS; High-resolution mass spectrometry; Bioanalytical method validation; Pharmacokinetics; Therapeutic drug monitoring; Pharmaceutical analysis