Work

Dual LC column characterization for mass spectrometry-based small molecule profiling of human plasma and serum

Year: 2025

Type: article

Abstract: Analyte annotation confidence in untargeted liquid chromatography mass-spectrometry (LC-MS) based chemical analysis can be enhanced by leveraging retention time information. For this, the chromatographic characteristics of the analytical system used should be well characterized. In this study, we measured 604 diverse chemical standards to characterize a dual LC setup consisting of pentabromobenzyl (PBr) and type-C silica hydride (SiH) columns operating in reversed-phase (RP) and aqueous normal-phase (ANP) mode, respectively. ANP and RP separations individually retained 40 % and 64 % of standards in cLogP range from −6.60 to 8.67 and −3.34 to 12.95, respectively. Using both columns, the coverage increased to 79 % of standards with cLogP range from −6.60 to 12.95 (median cLogP = 1.63). Retention selectivity follows the number of basic nitrogen atoms in the molecule on SiH column and polarity (cLogP) on PBr column. Column repeatability and reproducibility were tested in triplicate using a chemically diverse subset of 108 standards. Repeatability of retention times, peak widths and peak areas was 0.3 %, 14 %, 4 % for SiH column and 0.2 %, 12 %, 4 % for PBr column. Similarly, reproducibility was 15 %, 34 %, 30 % for SiH column and 9 %, 18 % and 34 % for PBr column. Predictive RT models were developed based on experimental RT data, achieving R 2 values of 0.92 and 0.96, with mean absolute errors of 0.29 min and 0.27 min for SiH and PBr columns, respectively. As proof of concept, 129 metabolites were annotated in pooled human serum and plasma by matching standard or predicted RT on one or both columns. The RT models and MS2 spectra of standards are openly available, facilitating uptake of this well-characterized chromatographic system to increase confidence in analyte annotation. • Silica hydride (SiH) & pentabromobenzyl (PBr) 2LC-MS retained 79 % of 604 chemicals. • Chemicals with clogP −6.60 to 12.95 were retained via the dual chromatography setup. • Predictive retention time models had R 2 > 0.9 & mean absolute error <0.3 min. • Raw data, predictive RT models and MS2 libraries are shared (CC BY-NC).

Source: Analytica Chimica Acta

DOI: 10.1016/j.aca.2025.343942

Authors: Žiga Tkalec, Štěpán Koudelka, Jana Klánová, Elliott J. Price

Institutions: Masaryk University, Jožef Stefan Institute, RECETOX

Language: English

Topic: Metabolomics and Mass Spectrometry Studies

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,270