Normal-Phase HPLC-ELSD to Compare Lipid Profiles of Different Wheat Flours

Abstract

Normal-phase high-performance liquid chromatography (HPLC) is widely used in combination with evaporative light scattering detection (ELSD) for separating and detecting lipids in various food samples. ELSD responses of different lipids were evaluated to elucidate the possibilities and challenges associated with quantification by means of HPLC-ELSD. Not only the number and type of polar functional groups but also the chain length and degree of unsaturation of (free or esterified) fatty acids (FAs) had a significant effect on ELSD responses. Tripalmitin and trilinolein yielded notably different ELSD responses, even if their constituting free FAs produced identical responses. How FA structure impacts ELSD responses of free FAs is thus not predictive for those of triacylglycerols and presumably other lipids containing esterified FAs. Because ELSD responses of lipids depend on the identity of the (esterified) FA(s) which they contain, fully accurate lipid quantification with HPLC-ELSD is challenging and time-consuming. Nonetheless, HPLC-ELSD is a good and fast technique to semi-quantitatively compare the levels of different lipid classes between samples of comparable FA composition. In this way, lipid profiles of different flours from near-isogenic wheat lines could be compared.

In vitro anti‐obesity activity by pancreatic lipase inhibition ‐ Simple HPLC approach using EVOO as natural substrate

“…The NP mode of elution, suitable for non‐mass selective detection, was selected to simultaneously separate major classes of lipids generated by the PL hydrolysis 24 . Indeed, differently from reversed phase, NP systems are particularly useful for lipid separation into individual class molecular species as a single peak, according to the number and type of polar functional groups 19,25 . Preliminary separation attempts were made by using different percentages of pure n ‐hexane and 2‐propanol, screening several gradient programs.…”

“…24 Indeed, differently from reversed phase, NP systems are particularly useful for lipid separation into individual class molecular species as a single peak, according to the number and type of polar functional groups. 19,25 Preliminary separation attempts were made by using different percentages of pure n-hexane and 2-propanol, screening several gradient programs. All the tested conditions did provide a co-elution between TAG and FFA peaks or between FFA and DAG peaks and were characterized by not reproducible retention times.…”

Lipid Signaling During Gamete Maturation

“…However, the improved methodology of high-performance thin-layer chromatography (HPTLC) still provides a powerful analytical tool to study lipid metabolism ( Fuchs et al, 2011 ). ELSD is also sensitive to the number and type of polar functional groups in different lipids as well as to fatty acid chain length and degree of unsaturation ( Melis et al, 2021 ). Furthermore, stable metabolic labeling studies to assess lipid signaling may also be fraught with technical difficulties especially in the absence of controls showing equilibrium labeling of the particular precursor pool of interest.…”

Quantification of Lipid and Peptide Content in Antigenic Peptide-loaded Liposome Formulations by Reversed-phase UPLC using UV Absorbance and Evaporative Light Scattering Detection

“…Increasing volumes (1,5,10,20,30, 40 and 50 µl) of DOTAP, DOPC and peptide stock solutions were injected by the autosampler of the UPLC system. Both DOTAP and DOPC stock solutions were 50 µg/ml in 1:1 MeOH:MQ (v/v) and the peptides (table 1, section 2.9) were dissolved in 0.04% (w/v) NH 4 OH or in CHCl 3 :MeOH:MQ water (60:36:4, v/v).…”

“…In general ELSD responses are nonlinear due to a correlation in-between solute concentration and aerosol particle size distribution upon nebulization and evaporation. The ELSD responses are therefore fitted by non-linear regression [20][21][22] .…”