Fluorescence probe assisted post-column detection for lipid analysis in microbore-LC

Area of Science:

• Analytical Chemistry
• Lipidomics
• Chromatography

Background:

• Lipid analysis often relies on destructive or less sensitive detection methods.
• Existing microbore-LC systems require specialized equipment for sensitive detection.
• Non-covalent fluorescence detection for lipids is underexplored, especially with microbore-LC.

Purpose of the Study:

• To develop and optimize a novel non-covalent fluorescence detection method for various lipid classes.
• To couple this method with non-aqueous reversed-phase microbore-LC (C18).
• To demonstrate the sensitivity, linearity, and versatility of the developed system.

Main Methods:

• Development of post-column fluorescence detection using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe.
• Adaptation of a classical LC system to microbore-chromatography (1 mm ID) without miniaturizing solvent delivery or detection.
• Optimization of detection parameters (probe concentration, flow rate, reactor length, temperature) using a central composite design (CCD) with phosphatidylcholine (PC) as a model lipid.

Main Results:

• Optimal detection conditions established: 3.35 µmol/L DPH, 0.5 mL/min post-column flow, 1.4 m reactor, 35°C.
• Linear fluorescence response for PC species from 5 µg/mL to 100 µg/mL.
• Lower limit of detection (LOD) of ~1 µg/mL, comparable to evaporative light scattering detection (ELSD).
• Successful detection of triglycerides, ceramides, and glycosphingolipids.

Conclusions:

• The developed DPH fluorescence detection method is sensitive, easy to use, and non-destructive.
• It provides a linear response across various lipid classes, unlike ELSD.
• The system demonstrates versatility and potential for broad application in lipidomics and analytical chemistry.