Dual flow-path HPLC method for time-optimised quantification of physicochemically different metabolite classes in...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Chromatography

Background:

• Biomedical studies require analyzing diverse analytes with varying properties.
• Single analytical methods often struggle to detect multiple analytes efficiently.
• Current multi-method approaches are time-consuming.

Purpose of the Study:

• To develop a time-optimized liquid chromatography-mass spectrometry (LC-MS) method.
• To enable parallel analysis of short-chain fatty acids (SCFA) and amines from human serum.
• To reduce overall sample analysis time while maintaining analytical performance.

Main Methods:

• Utilized a single mass spectrometer with parallel hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP)-HPLC.
• Employed a six-port valve and dual split sampler for simultaneous column re-equilibration.
• Derivatized SCFA for improved RP-HPLC separation and electrospray ionization; analyzed amines in native form via HILIC.

Main Results:

• Achieved a 37% reduction in analysis time compared to individual methods.
• Demonstrated robust and reproducible quantification in human serum samples (RSD ≤ 5.8%).
• Maintained stable retention times and chromatographic resolutions for both SCFA and amine analyses.

Conclusions:

• The parallel RP-HPLC and HILIC approach with a single MS is highly efficient.
• This method allows customized chromatographic conditions for extensive sample batches.
• Optimized detector utilization and separation performance for diverse analytes.