Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes.

Autor:	Zhang NR; Department of Discovery, Preclinical and Translational Medicine, Merck & Co., Inc., West Point, PA, USA. Electronic address: rena_zhang@merck.com., Hatcher NG; Department of Neuroscience, Merck & Co., Inc., West Point, PA, USA. Electronic address: Nathan_hatcher@merck.com., Ekroos K; Lipidomics Consulting Ltd, Esbo, Finland., Kedia K; Department of Discovery, Preclinical and Translational Medicine, Merck & Co., Inc., West Point, PA, USA., Kandebo M; Department of Neuroscience, Merck & Co., Inc., West Point, PA, USA., Marcus JN; Department of Neuroscience, Merck & Co., Inc., West Point, PA, USA., Smith SM; Department of Neuroscience, Merck & Co., Inc., West Point, PA, USA., Bateman KP; Department of Discovery, Preclinical and Translational Medicine, Merck & Co., Inc., West Point, PA, USA., Spellman DS; Department of Discovery, Preclinical and Translational Medicine, Merck & Co., Inc., West Point, PA, USA.
Jazyk:	angličtina
Zdroj:	Journal of lipid research [J Lipid Res] 2022 Jun; Vol. 63 (6), pp. 100218. Date of Electronic Publication: 2022 Apr 27.
DOI:	10.1016/j.jlr.2022.100218
Abstrakt:	A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery. Competing Interests: Conflicts of interest The authors declare that they have no competing interests on every aspect of the work. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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