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

A general approach, still few exploited so far and never associated with microbore-LC, consisting of detection of various lipid classes (i.e. phospholipids, triglycerides, ceramides and glycosphingolipids) by non-covalent association with 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence probe is developed. This mode of detection was coupled with non-aqueous reversed-phase microbore-LC (C18) by using classical post-column fluorescence detection. The classical LC system was first adapted to microbore-chromatography (internal diameter 1 mm) without apparatus miniaturization of the solvent delivery system and the detection cell. For this purpose, the detection parameters (probe concentration, post-column flow rate, post-column reactor length and post-column system temperature) were optimized by a central composite design (CCD) using a mixture of phosphatidylcholine (PC) species as a lipid model and DPH (λex = 350 nm, λem = 430 nm) as a fluorescence probe. The optimal conditions of detection for the various molecular species of PC were determined for a DPH concentration of 3.35 μmol/L, a post-column flow rate of 0.5 mL/min, a reactor length of 1.4 m and a temperature of 35 °C. The fluorescence response was linear over a wide range of PC species from 5 μg/mL to 100 μg/mL and the lower limit of detection (signal/noise = 3) was about 1 μg/mL, that is equivalent to evaporative light scattering detection (ELSD). Others molecular species of various classes of lipids, i.e. triglycerides, ceramides and glycosphingolipids were also easily detected. Thus, this study demonstrated the versatility of the proposed system of detection which was shown to be sensitive, easy to perform, non-destructive and allowed, in contrast to ELSD, for a linear response with various polarity lipid classes.