Enhancing coverage in LC-MS-based untargeted metabolomics by a new sample preparation procedure using mixed-mode...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Metabolomics

Background:

• Untargeted metabolomics using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) faces challenges in metabolome coverage.
• Limited coverage stems from weak signals of hydroxyl and carboxyl groups and LC separation limitations for diverse polarities.

Purpose of the Study:

• To develop a novel sample preparation procedure to enhance metabolome coverage in LC-MS-based untargeted metabolomics.
• To improve detection limits and identify a broader range of metabolites, including those with weak ESI signals.

Main Methods:

• Utilized mixed-mode solid-phase extraction to separate metabolites into hydrophilic amine, hydrophobic amine/alcohol, and organic acid groups.
• Employed pyridine derivatization for signal enhancement of alcohols and carboxylic acids.
• Analyzed hydrophilic amines using hydrophilic interaction LC-MS and hydrophobic compounds using C18 LC-MS.

Main Results:

• Achieved significantly lower detection limits for amino acids (3.3–70x) and fatty acids (65–1141x) compared to traditional methods.
• Detected cholesterol with a 125 ng mL⁻¹ detection limit, a significant improvement over the classic method.
• Identified 110 additional metabolites in seminal plasma samples, including 53 carboxylic acids and 21 alcohols unique to the new method.

Conclusions:

• The new sample preparation method substantially expands the metabolome coverage of LC-MS-based untargeted metabolomics.
• Demonstrated increased identification of hydrophilic amines, carboxylic acids, and alcohols.
• Showcased potential in identifying biomarkers for male infertility, with 46 potential biomarkers found compared to 19 by the classic method.