Structure elucidation and mitigation of endogenous interferences in LC-MS-based metabolic profiling of urine

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Title: Structure elucidation and mitigation of endogenous interferences in LC-MS-based metabolic profiling of urine
Authors: Albreht, A
Hussain, H
Jimenez, B
Armstrong, A
Whiley, L
Witt, M
Lewis, M
Chekmeneva, E
Item Type: Journal Article
Abstract: Liquid chromatography mass spectrometry (LC-MS) is the main workhorse of metabolomics owing to its high degree of analytical sensitivity and specificity when measuring diverse chemistry in complex biological samples. LC-MS-based metabolic profiling of human urine, a biofluid of primary interest for clinical and biobank studies, is not widely considered to be compromised by the presence of endogenous interferences and is often accomplished using a simple “dilute-and-shoot” approach. Yet, it is our experience that broad obscuring signals are routinely observed in LC-MS metabolic profiles and represent interferences which lack consideration in the relevant metabolomics literature. In this work we chromatographically isolated the interfering metabolites from human urine and unambiguously identified them via de novo structure elucidation as two separate proline-containing dipeptides: N,N,N-trimethyl-L-alanine-L-proline betaine (L,L-TMAP) and N,N-dimethyl-L-proline-L-proline betaine (L,L-DMPP), the latter reported here for the first time. Offline LC-MS/MS, MRMS, and NMR spectroscopy were essential components of this workflow for the full chemical and spectroscopic characterization of these metabolites and for establishing the co-existence of cis and trans isomers of both dipeptides in solution. Analysis of these definitive structures highlighted intramolecular ionic interactions as responsible for slow interconversion between these isomeric forms resulting in their unusually broad elution profiles. Proposed mitigation strategies, aimed at increasing the quality of LC-MS-based urine metabolomics data, include modification of the column temperature and mobile phase pH to reduce the chromatographic footprint of these dipeptides, thereby reducing their interfering effect on the underlying metabolic profiles. Alternatively, sample dilution and internal standardization methods may be employed to reduce or account for the observed effects of ionization suppression on the metabolic profile.
Issue Date: 13-Jan-2022
Date of Acceptance: 7-Dec-2021
DOI: 10.1021/acs.analchem.1c04378
ISSN: 0003-2700
Publisher: American Chemical Society
Journal / Book Title: Analytical Chemistry
Volume: 94
Issue: 3
Sponsor/Funder: Medical Research Council
Medical Research Council
National Institute for Health Research (NIHR)
Funder's Grant Number: MR/S010483/1
Keywords: Science & Technology
Physical Sciences
Chemistry, Analytical
Analytical Chemistry
0301 Analytical Chemistry
0399 Other Chemical Sciences
Publication Status: Published online
Appears in Collections:Department of Metabolism, Digestion and Reproduction
Department of Surgery and Cancer
Faculty of Medicine

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