Enhancing metabolite coverage using dedicated mobile phases for individual polarity modes in HILIC-MS

Area of Science:

• Analytical Chemistry
• Biochemistry
• Metabolomics

Background:

• Metabolomic studies require sensitive and comprehensive analytical methods for accurate profiling.
• Existing hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) methods may have limitations in metabolite coverage and identification confidence.
• Optimization of mobile phases and chromatographic systems is crucial for enhancing HILIC-MS performance.

Purpose of the Study:

• To develop and optimize a novel HILIC-MS method for enhanced metabolite coverage and identification confidence.
• To investigate the impact of mobile phase composition, pH, and additives on MS response in a bioinert system.
• To evaluate the performance of the optimized HILIC-MS method in complex biological matrices.

Main Methods:

• Development of a dual mobile phase strategy for HILIC-MS.
• Optimization of mobile phase composition, pH, and evaluation of chelating additives.
• Utilizing a bioinert chromatographic system and a bridged ethyl hybrid (BEH) amide column.
• Application of the optimized method to analyze mouse plasma, pancreas, and liver samples.

Main Results:

• The dual mobile phase strategy significantly expanded the range of annotated metabolites and improved identification confidence.
• Alkaline pH was critical for sensitive detection of polyphosphorylated metabolites.
• A bioinert system rendered chelating additives redundant.
• The BEH amide column demonstrated superior performance for metabolomic analysis.
• The method showed reliable performance in complex biological samples.

Conclusions:

• The developed HILIC-MS method provides broader and more accurate metabolic profiles with enhanced confidence.
• The optimized method is versatile and reliable for metabolomics studies in complex biological matrices.
• This technique represents a powerful tool for confident and reproducible metabolomics research.