Key Points:
- Advances in automated, high-throughput workflows are streamlining method development by enabling rapid exploration of complex variable spaces, improving efficiency and reproducibility in both research and industrial settings.
- As incremental MS improvements become costlier, customized LC systems—including online preconcentration and high-volume injections—are emerging as more impactful tools for improving sensitivity in drug metabolism and trace analysis of complex matrices.
- The application of LC–MS-based MAM for nanobody quality control underscores a trend toward replacing traditional HPLC–UV with more data-rich, multiplexed analyses that improve product characterization and facilitate regulatory compliance through smarter data reuse and risk analysis.
- Microsampling and microflow LC–MS/MS are significantly improving the ethical and statistical quality of preclinical pharmacokinetic studies by reducing animal use, increasing sensitivity, and uncovering meaningful biological variation that pooled sampling might obscure.
Day 2 of HPLC 2025 on Tuesday, June 17, 2025, ended with an illuminating session on pharmaceutical separations chaired by Debby Mangelings and Todd Maloney.
Erik Regalado from Merck & Co. started the session with a presentation entitled Automated Multicolumn Screening Workflow in Ultra-High Pressure HILIC Chromatography for Streamlined Method Development of Polar Analytes. Regalado discussed advances in hydrophilic interaction chromatography (HILIC) for analyzing polar compounds, highlighting an automated workflow designed to streamline method development. Traditional HILIC optimization can be tedious due to the many interacting variables involved. Regalado introduced a high-throughput screening method using 12 different ultrahigh-pressure liquid chromatography (UHPLC)-compatible columns, including both fully and superficially porous particles, tested across a wide pH range and with multiple organic solvents. The system integrates diode array, charged aerosol, and mass spectrometry detection to enhance analytical flexibility. This platform facilitates rapid and reliable assay development, offering a standardized and efficient path for identifying optimal HILIC conditions in both research and industrial contexts, according to Regaldo.
Enhancing Sensitivity in Drug Metabolism Studies with Cutting-Edge LC Configurations was presented by Filip Cuyckens of Johnson & Johnson, Belgium. Cuyckens emphasized the importance of maximizing sensitivity in drug metabolism research, particularly as sample volumes decrease and the demand for precision increases. While advances in mass spectrometry have contributed to improved detection, these gains are becoming increasingly marginal and costly. Instead, Cuyckens presented custom-designed liquid chromatography (LC) setups tailored to specific sample sizes and detectors, significantly enhancing sensitivity. Case studies included the use of online preconcentration and injection volumes ranging from 80 µL to 500 µL for various analytical techniques, such as LC–tandem mass mass spectrometry (MS/MS) and LC/fluor-ICP-MS. He concluded that these innovations allow for better utilization of limited samples and improved analysis of complex biological matrices. Cuyckens concluded that the LC/fluor-ICP-MS setup is a promising alternative for radiolabeled human metabolism studies.
Multi-Attribute Monitoring for QC Release Testing of a Therapeutic Nanobody was presented by Gwenael Nys from Sanofi in Geel, Belgium. Nys described Sanofi’s development of a mass spectrometry-based multi-attribute monitoring (MAM) method for quality control (QC) testing of therapeutic nanobodies. MAM, using LC–MS, enables simultaneous tracking of multiple product quality attributes (PQAs), offering detailed insights into protein variants and enhancing the reusability of historical data. This method is positioned as a more informative and efficient alternative to traditional high performance liquid chromatography (HPLC)–UV techniques. Nys outlined key aspects of method development, including optimization of chromatographic and mass spectrometric conditions, as well as sample handling. He then detailed the challenges and considerations involved in transferring this approach into a GMP-compliant QC setting, including method validation, risk analysis, and bridging with legacy assays. Nys emphasized that the MAM method enhances understanding of product modifications while maintaining regulatory compliance, proposing it as a transformative tool in biopharmaceutical QC environments.
A talk with a rather long title—Going Micro: Pharmacokinetic Insulin Profiles Obtained from Microsampled Rat Plasma and a Microflow LC–MS/MS Assay Result in Higher Sensitivity and Statistical Power, Less Trauma Imposed on Animals, and Fewer Animals Used Compared to a Conventional Approach—was presented by Nikoline Juul Nielsen on behalf of a team from the University of Copenhagen and Novo Nordisk, Denmark. Nielsen discussed how microsampling combined with a microflow LC–MS/MS assay can significantly improve pharmacokinetic (PK) analysis in preclinical insulin studies. Unlike traditional sparse sampling, which requires pooling data from multiple animals, microsampling enables complete PK profiles from individual rodents, reducing animal use and stress in line with the 3Rs (Replacement, Reduction, Refinement). Nielsen highlighted how a miniaturized LC–MS/MS setup (1 µL/min flow, 0.1 mm i.d.) achieved a 47-fold sensitivity increase compared to conventional systems, allowing quantification of insulin degludec in very low plasma volumes. Despite reduced sample preparation, results from micro- and conventional samples were consistent (p = 0.89). Importantly, individual profiles revealed significant inter-animal variation (p = 0.0023), demonstrating that pooled data may misrepresent treatment effects. The study advocates for broader adoption of microsampling in PK studies to improve data quality and uphold ethical standards in animal research, according to Nielson
