Best of the Week: Statistical Practices in Chromatography, Glycoform Characterization of Monoclonal Antibodies

This past week, LCGC International published a variety of articles on hot topics in separation science. We discuss the importance of sound statistical practices in chromatography, highlighting two recent columns from Bob W. J. Pirok from the University of Amsterdam, a new interview about glycoform characterization of monoclonal antibodies, and a recent study that detected ivermectin (IVM) residues in intensive swine farms using LC-MS/MS.

This is the Best of the Week.

Behind the Peaks: The Analyst's Dilemma

In this episode of “Go Behind the Peaks” from LCGC International, we explore the importance of sound statistical practices in chromatography. Drawing from two articles written by Bob W. J. Pirok, the podcast emphasizes that limited replicates undermine statistical confidence and that more repetitions improve accuracy and reliability (1). There is also a discussion about the value of using robust statistics like the median and median absolute deviation (MAD) to handle outliers effectively (1). The episode highlights the value of data visualization tools such as box plots to reveal patterns and enhance reproducibility in chromatographic and machine learning applications (1).

Enhanced Intact Glycoform Characterization of Monoclonal Antibodies Using Polyacrylamide Monolithic HILIC-MS

In an interview with LCGC International, Annika van der Zon and Andrea Gargano from the van ’t Hoff Institute for Molecular Sciences discussed their development of poly(acrylamide-co-N,N-methylene-bis(acrylamide)) monolithic HILIC columns for improved intact glycoprotein separation. These polymer-based monoliths, featuring non-charged surfaces and optimized pore morphology, outperform conventional silica phases by minimizing ionic interactions and enhancing resolution at lower trifluoroacetic acid (TFA) levels (2). The researchers addressed challenges in separating large IgG monoclonal antibody (mAb) glycoforms, whose tiny mass differences complicate resolution (2). By carefully adjusting DMSO-to-octanol ratios during polymerization, they achieved better mass transfer, narrower peaks, and the first LC–MS method capable of distinguishing intact mAb glycoforms (2).

Hydrophilic Interaction Chromatography for the Analysis of Therapeutic Oligonucleotides

In an interview with LCGC International, Davy Guillarme discussed advances in hydrophilic interaction chromatography–mass spectrometry (HILIC–MS) for analyzing therapeutic oligonucleotides and their impurities. Although ion-pairing reversed-phase liquid chromatography (IP-RPLC) remains the gold standard because of its selectivity and regulatory acceptance, HILIC offers simpler MS compatibility by avoiding ion-pairing reagents (3). Challenges include adsorption on metal surfaces, limited selectivity, and reduced MS sensitivity from alkali adducts (3). Guillarme emphasized using bioinert columns and optimizing mobile phases to improve performance (3). His next research project aims to refine HILIC conditions by exploring how alternative solvents and additives can enhance oligonucleotide impurity separation and reduce ion adduct formation.

Analyzing Small RNA-based Therapeutics and Their Process Impurities Using Fast and Sensitive LC–HRMS

Silvia Millán-Martín of the National Institute for Bioprocessing Research and Training (NIBRT) discusses her team’s development of an ion-pair reversed-phase liquid chromatography–high-resolution mass spectrometry (IP-RPLC–HRMS) method for characterizing small RNA-based therapeutics (4). Using a novel pentylamine–HFIP combination and a mild heated electrospray ionization (H-ESI) source, the method minimizes adduct formation and in-source artifacts while maintaining sensitivity and robustness (4). This approach enables accurate profiling of impurities and sequence variants in antisense oligonucleotides, supporting regulatory compliance and quality control (4). By balancing chromatographic resolution and MS sensitivity, it offers a versatile, eco-friendly workflow to accelerate the development and approval of RNA-based drugs.

Quantifying Ivermectin in Swine Farm Environments Using LC-MS/MS

A recent study by the University of Parma and the Experimental Zooprophylactic Institute of Lombardy and Emilia Romagna detected ivermectin (IVM) residues in intensive swine farms using LC–MS/MS. This study, which was published in Veterinary Medicine and Science, showed that the administration route resulted in varying fecal concentrations, with orally treated sows initially showing higher levels (5). Soil fertilized with contaminated slurry retained IVM residues exceeding ecotoxicological thresholds for non-target species like dung beetles and earthworms (5). The study underscores potential environmental risks of veterinary drug use in livestock and calls for improved management practices and further research to refine environmental safety guidelines.

References

1. Jones, K. Behind the Peaks: The Analyst's Dilemma. LCGC International. Available at: https://www.chromatographyonline.com/view/behind-the-peaks-the-analyst-s-dilemma (accessed 2025-10-29).
2. Chasse, J. Enhanced Intact Glycoform Characterization of Monoclonal Antibodies Using Polyacrylamide Monolithic HILIC-MS: Part II. LCGC International. Available at: https://www.chromatographyonline.com/view/enhanced-intact-glycoform-characterization-of-monoclonal-antibodies-using-polyacrylamide-monolithic-hilic-ms-part-ii (accessed 2025-10-29).
3. Chasse, J. Hydrophilic Interaction Chromatography for the Analysis of Therapeutic Oligonucleotides. LCGC International. Available at: https://www.chromatographyonline.com/view/hydrophilic-interaction-chromatography-for-the-analysis-of-therapeutic-oligonucleotides (accessed 2025-10-29).
4. Matheson, A. Analyzing Small RNA-based Therapeutics and Their Process Impurities Using Fast and Sensitive LC–HRMS. LCGC International. Available at: https://www.chromatographyonline.com/view/analyzing-small-rna-based-therapeutics-and-their-process-impurities-using-fast-and-sensitive-lc-hrms (accessed 2025-10-29).
5. Chasse, J. Quantifying Ivermectin in Swine Farm Environments Using LC-MS/MS. LCGC International. Available at: https://www.chromatographyonline.com/view/quantifying-ivermectin-in-swine-farm-environments-using-lc-ms-ms (accessed 2025-10-29).