Study of the analysis of alkoxyglycerols and other non-polar lipids by liquid chromatography coupled with evaporative light scattering detector

Abstract

An HPLC method with evaporative light scattering detection (ELSD) for the simultaneous analysis of various lipid classes, particularly alkoxyglycerols and acylglycerols with very similar structure and polarity, has been developed. These lipid classes are frequently found in numerous fats and oils such as shark liver oils and can serve as substrates for lipase-catalyzed reactions. This method utilizes a silica column and a gradient elution of isooctane, methyl tert-butyl ether and 2-propanol in different proportions. Separation between squalene, sterol esters, and fatty acid ethyl esters has been achieved in a time of analysis slightly higher than 8 min. In addition, a good resolution between 1,3-diacylglycerols and free sterols was also attained in the same run, with a broad range of concentrations. Excellent precision regarding the retention times was obtained. The limit of detection for the different lipid classes studied was below 1 μg. Intra-day and inter-day variation of retention times and areas was also evaluated. The relative standard deviation of intra-day variation for retention times and areas never exceeded of 0.1 and 10, respectively. The HPLC-ELSD method was also optimized to separate and quantify the hydrolysis products of alkoxyglycerols and acylglycerols (mono-esterified and non-esterified alkoxyglycerols and mono-esterified and di-esterified acylglycerols) at the same time, rendering a useful method for the study of lipase-catalyzed reactions and a wide variety of fats and oils. The present methodology not only separates 18 different lipid classes with a good reproducibility, but it is also able to estimate the relative proportion in which they are found in a broad range of concentrations.