Evaluation of ion source parameters and liquid chromatography methods for plasma untargeted metabolomics using orbitrap mass spectrometer

Authors: ASSRESS, HAILEMARIAM - Arkansas Children'S Nutrition Research Center (ACNC); FERRUZZI, MARIO - Arkansas Children'S Nutrition Research Center (ACNC); HAMEED, AHSAN - Arkansas Children'S Nutrition Research Center (ACNC); PACK, LINDSAY - Arkansas Children'S Nutrition Research Center (ACNC); LAN, RENNY - Arkansas Children'S Nutrition Research Center (ACNC)

Submitted to: The Journal of Chromatography B: Biomedical Applications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/11/2025
Publication Date: 3/19/2025
Citation: Assress, H.A., Ferruzzi, M.G., Hameed, A., Pack, L.M., Lan, R.S. 2025. Evaluation of ion source parameters and liquid chromatography methods for plasma untargeted metabolomics using orbitrap mass spectrometer. The Journal of Chromatography B: Biomedical Applications. 1257(2025). Article 124564. https://doi.org/10.1016/j.jchromb.2025.124564.
DOI: https://doi.org/10.1016/j.jchromb.2025.124564

Interpretive Summary: Metabolomics, the systematic measurements of metabolites in biological matrices, utilizing liquid chromatography(LC) coupled with high-resolution mass spectrometry (MS), has become trendy and demonstrated its enormous promise and power in the study of health and disease. Comprehensive high-throughput untargeted metabolomics requires development of sensitive and reliable analytical methods. In this work, Electrospray ionization (ESI) parameters and spray probe positions were evaluated and optimized for maximal signal intensity and higher number of compound detection. Different class of metabolites were also evaluated in relation to the ESI parameters. In addition, we present a comparison of chromatographic conditions containing different mobile phase gradients, five reversed phase (RP)-C18 column and two hydrophilic interaction liquid chromatography (HILIC) columns for untargeted metabolomics. combining the optimal RP and HILIC approaches greatly expanded metabolome coverage with 60% new metabolic features detected compared with RP column alone for serum samples. These findings confirm that ESI parameters and chromatographic conditions do influence metabolomics results, and propose strategies for increasing metabolite coverage in untargeted metabolomics.

Technical Abstract: The method used in the analytical step heavily determines the sensitivity, selectivity, metabolome coverage, reproducibility, rate of false positive and/or false negative discovery and throughput of the untargeted metabolomics. Comprehensive and high-throughput untargeted metabolomics necessitate the development and validation of reproducible and reliable analytical methods to reduce any analytical biases. In this work, we explored the influence of electrospray ionization (ESI) needle position and parameters including the positive ion spray voltage, negative ion spray voltage, sheath gas, auxiliary gas, ion transfer tube (ITT) temperature (°C), and vaporization temperatures (°C) in metabolite annotations. In addition, we present a comparison of chromatographic conditions containing different mobile phase gradients, five reversed phase (RP)-C18 column and two hydrophilic interaction liquid chromatography (HILIC) columns for untargeted metabolomics. Our results revealed that ESI needle position, fraction of organic solvent and the ESI parameters affected notably the signal stability and hence the number of metabolite annotations. Despite the slight differences in chromatographic characteristics, the RP columns premier CSH C-18, HSS T3 C-18, and BEH C-18 showed comparable performance in terms of number of metabolite annotations. Among the HILIC columns tested, the zwitterionic BEH Z-HILIC demonstrated better suitability for untargeted metabolomics. Finally, combining the optimal RP and HILIC approaches greatly expanded metabolome coverage with 60% new metabolic features detected compared with RP column alone for serum samples. These findings confirm that ESI parameters and chromatographic conditions do influence metabolomics results, and propose strategies for increasing metabolite coverage in untargeted metabolomics. A limitation of this work is that our parameters were only optimized for a single matrix on Orbitrap mass spectrometer and may be different for other matrix and other mass spectrometer platforms.