Study of the analysis of alkoxyglycerols and other non-polar lipids by liquid chromatography coupled with evaporative light scattering detector
Carlos F. Torres, Luis V´ zquez, F. Javier Se˜ or´ ns, Guillermo Reglero ∗ a n a
Secci´ n Dept. de Ciencias de la Alimentaci´ n, Facultad de Ciencias, Universidad Aut´ noma de Madrid, Cantoblanco, 28049Madrid, Spain o o o Received 9 December 2004; received in revised form 31 March 2005; accepted 6 April 2005
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 innumerous 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, agood 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 g. Intra-day and inter-day variation of retention times and areas was also evaluated. The relative standard deviation of intra-dayvariation 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 reactionsand 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. © 2005 Elsevier B.V. All rights reserved.
Keywords: Alkoxyglycerols; ELSD; HPLC; Normal-phase; Non-polar lipids
1. Introduction Several HPLCmethods for the analysis of lipid classes have been described. Some of these methods utilize cyanopropyl columns for the separation of lipid classes . Nine lipid classes were also separated on a dihydroxypropyl column . Up to 12 different lipid classes have been simultaneously analyzed using normal stationary phase enriched in hydroxyl groups, namely diol columns . All these methods utilizeevaporative light scattering detectors (ELSD) which have brought a major advance in the detection of lipid classes by HPLC. This detector is not limited by the nature of the solvent, ﬂow rate, or ambient temperature. In addition, with
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ELSD no lipid derivatization isrequired and provides a quantitative response [4,5]. One of the difﬁculties of these normal-phased HPLC methods is the simultaneous separation of hydrocarbons, such as squalene, sterol esters, and fatty acid methyl or ethyl esters combined with a good resolution of more polar lipids in the same run. Some methods have been described for the separation of non-polar lipid classes using alumina asstationary phase [6,7]. Schaefer et al. have successfully separated wax esters, sterol esters, fatty acid methyl esters on a diol column although peak splitting of lipid classes was observed mostly due to the different degrees of saturation of the fatty acid residues coupled to the lipid. In the present study we have developed an HPLC method for the analysis of non-polar lipids. In particular, we...