The second part of the end-of-the-year review of LCGC International’s editorial coverage of HPLC 2025, which took place in Bruges June 19–23, 2025.

A major highlight in the chromatography conference calendar this year was without a doubt HPLC 2025, which was held from June 19 to 25 in Bruges, Belgium, with Gert Desmet, Deirdre Cabooter, Sebaastian Eeltink, and Ken Broeckhoven at the helm of the organizing committee. Under the all-seeing eye of Pat Sandra from the RIC group, HPLC 2025 exemplified why face-to-face interaction remains so essential in the chromatography community. Separation science has been gradually evolving, driven by technological developments, a focus on sustainable separation science practices, advances in artificial intelligence (AI) and machine learning (ML), and shifting regulatory expectations. It is important to stay informed by reading the latest peer-reviewed articles, technical articles, interviews, opinion pieces, and attending virtual events, but being part of the conversation as it happens “in reality” stimulates creativity in the separation science community. LCGC International provided a snapshot of some of the highlights of this year’s event.

Modern Method Development The session focused on the growing role of data-driven and AI-assisted approaches in modern analytical chemistry to help address the complexity, time, and cost of method development. Emphasis was placed on combining mechanistic understanding with machine learning and optimization tools to handle interdependent parameters and reduce experimental workload. Advances in instrumentation, modeling, and workflow design were highlighted as enabling more robust, predictive, and automated analyses across diverse applications. Overall, the theme underscored a shift toward integrated, intelligent analytical systems that augment expert knowledge rather than replace it.

Peter Schoenmakers (University of Amsterdam, Amsterdam, Netherlands) highlighted the complexity and cost of liquid chromatography (LC) method development, particularly for two-dimensional LC (LC×LC), where optimization can take months. He emphasized the need for computer-assisted and AI-supported tools to handle interdependent parameters, while noting that expert knowledge remains crucial.

Kevin Schug (University of Texas at Arlington, Texas, USA) demonstrated how data science, machine learning, and surrogate optimization can streamline analytical method development. Using supercritical fluid extraction–supercritical fluid chromatography (SFE–SFC) as an example, he showed that structure-based molecular feature encoding improves predictive performance and reduces experimental effort.

Gunda Koellensperger (University of Vienna, Austria) discussed challenges in ex vivo tissue metabolomics, stressing the importance of sampling strategies and control of analytical variables. Advances in high-resolution mass spectrometry (HRMS) and miniaturized workflows are enabling more reliable clinical metabolomics applications.

Debby Mangelings (Vrije Universiteit Brussel, Belgium) presented quantitative structure enantioselective retention relationship (QSERR) models for predicting chiral separations on polysaccharide-based phases. By combining achiral and chiral molecular descriptors, her approach accurately predicts enantiomer retention, selectivity, and elution order across diverse pharmaceuticals.

Fanyi Duanmu (University College London, United Kingdom) described an AI-driven “Smart HPLC Robot” that integrates mechanistic models, machine learning, and a digital twin to automate method development, significantly reducing experimental time and resource use.

José-Ramón Torres-Lapasió (University of Valencia, Spain) showed that global retention models effectively predict retention shifts in serially coupled columns, particularly under gradient conditions, supporting optimized hybrid column high performance liquid chromatography (HPLC) strategies.

Read more about this session here: Modern Method Development

Pharmaceutical Separation Science Session Day two of HPLC 2025 concluded with a session on pharmaceutical separations chaired by Debby Mangelings (Vrije Universiteit Brussel, Brussels, Belgium) and Todd Maloney (Eli Lilly, USA).

Erik Regalado (Merck & Co., New Jersey, USA) opened with a presentation on an automated multicolumn ultrahigh-pressure liquid chromatography–hydrophilic interaction chromatography (UHPLC–HILIC) screening workflow for polar analytes. He described a high-throughput approach using 12 UHPLC-compatible columns, spanning wide pH ranges and organic solvents, and incorporating diode array, charged aerosol, and mass spectrometry detection. This platform simplifies the traditionally complex HILIC optimization process and enables rapid, standardized method development for research and industrial applications, according to Regaldo.

Filip Cuyckens (Johnson & Johnson, Belgium) followed with a talk on enhancing sensitivity in drug metabolism studies through optimized liquid chromatography configurations rather than incremental mass spectrometry upgrades. He presented custom LC setups using online preconcentration and large injection volumes (80–500 µL) for techniques including LC–MS/MS and LC/fluor-ICP-MS. Cuyckens concluded these approaches improve analysis of limited and complex biological samples, with LC/fluor-inductively coupled plasma (ICP)-MS highlighted as a promising alternative for radiolabeled metabolism studies.

Gwenael Nys (Sanofi, Belgium) presented the development of a mass spectrometry-based multi-attribute monitoring (MAM) method for QC release testing of therapeutic nanobodies. Using LC–MS, MAM allows simultaneous monitoring of multiple product quality attributes and provides greater insight than traditional HPLC–UV methods. Nys discussed method development, validation, and challenges of implementing MAM in a GMP-compliant QC environment.

The session concluded with a presentation by Nikoline Juul Nielsen (University of Copenhagen/Novo Nordisk, Denmark) on microsampling combined with microflow LC–MS/MS for insulin pharmacokinetic studies in rats. This approach demonstrated complete PK profiles from individual animals with a 47-fold sensitivity increase, reduced animal use, and revealed inter-animal variability that pooled sampling can obscure, supporting improved data quality and ethical research practices.

Martin Vollmer, biopharma program manager at Agilent Technologies, spoke to LCGC International about the evolving importance of glucagon-like peptide-1 (GLP-1) therapeutics and the challenges they present to separation scientists. Read the full interview here: The Rise of GLP-1 Therapeutics

Read more about this session here: Pharmaceutical Separation Science Session.

LCGC International Video Coverage Alasdair Matheson, executive editor of LCGC International, was on-site to conduct video interviews with some of the key opinion leaders in separation science, including:

Paul Ferguson, an active member of LCGC International’s editorial advisory board (EAB) and the Chromatographic Society (ChromSoc), is a leading voice in sustainability in the pharmaceutical sector. In this short video interview, he explored the intersection of green chemistry and emerging therapeutics: Sustainability and Emerging Therapeutics

Torgny Fornstedt, professor in analytical chemistry and leader of the Fundamental Separation Science Group (Karlstad University, Sweden), gave a detailed interview on the challenges and solutions involved in the analysis of oligonucleotides*. *View Part One and Part Two

Gesa Schad from Shimadzu Europe, Germany, discusses how SFC is being used in practice, and why she launched SFC For Dummies: SFC for Dummies Book Launch

**HPLC 2025, Essential Reading **As well as conference reportage and digital coverage, there was also essential editorial content organized in synchronicity with the event:

“Column Watch” editor, Dave Bell, delivered his annual “must-read” guide to the hottest topics at this year’s conference, including technological developments, industry trends, and prestige poster winners. Read the full review here: Highlights From HPLC 2025

LCGC International’s HPLC Companion 2025 also put a spotlight on important topics covered at the event, including Advances in Automation; Modern Non-targeted Screening Workflows; Sustainability and Separation Science; Going Beyond Micro-pillar Array Columns; Portable LC Instruments, The Evolution of 2D LC; and Functionalized Monoliths for Sample Preparation.

Gert Desmet from the Vrije Universiteit, Brussels, Belgium, guest-edited a special supplement related to HPLC 2025—Advances in (U)HPLC—with contributions from key speakers at the event. Topics included a combined iterative DDA–DIA mass-spectrometry workflow, the use of LC×SFC orthogonal 2D separations for complex samples, the role of exposomics in food-safety applications, non-animal approaches to predicting skin permeability using LC and SFC, the development of 3D-printed sorbents for untargeted LC–QTOF-MS, and high-throughput metabolomics using dried blood spots (DBS).

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