A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction

Abstract

Efficient isolation of individual lipid classes is a critical step in the analysis of plasma and lipoprotein fatty acid compositions. Whilst good separations of total lipid extracts are possible by TLC, this method is time consuming and a major rate-limiting step when processing large numbers of specimens. A method for rapid separation of phosphatidylcholine (PC), non-esterified fatty acids (NEFA), cholesterol ester (CE) and triacylglycerol (TAG) from total plasma lipid extracts by solid-phase extraction (SPE) using aminopropyl silica columns has been developed and validated. Following initial separation of polar and neutral lipids, individual classes were isolated by application of solvents with increasing polarity. Recoveries for combined plasma extraction with chloroform–methanol and SPE were (%): PC 74·2 (SD 7·5), NEFA 73·6 (sd 8·3), CE 84·9 (sd 4·9), and TAG 86·8 (sd 4·9), which were significantly greater for TAG and NEFA than by TLC (P<0·001). Both GC–flame ionisation detector and GC-MS analysis of fatty acid methyl esters demonstrated that there was no cross-contamination between lipid classes. Measurements of repeatability of fatty acid composition for TAG, PC, CE and NEFA fractions showed similar CV for each fatty acid. The magnitude of the CV appeared to be related inversely to the fractional fatty acid concentration, and was greatest at concentrations of less than 1 g/100 g total fatty acids. There was no evidence of selective elution of individual fatty acid or CE species. In conclusion, this method represents an efficient, rapid alternative to TLC for isolation of these lipid classes from plasma.

Effect of dietary marine lipids on female white bass ova compositions and progeny survival

“…Total lipid content was determined for lyophilized ova and diets using the method of Folch et al (1957). Ova lipid classes were then separated into PL, cholesteryl ester (CE), and TAG fractions using solid-phase extraction (see Burdge et al 2000). Total lipid and individual lipid classes were subjected to acid-catalyzed transmethylation performed overnight at 50°C following the method developed by Christie (1982).…”

Fatty Acid Profile of Sunshine Bass: II. Profile Change Differs Among Fillet Lipid Classes

“…Phospholipid (PL), cholesteryl ester (CE), and triacylglycerol (TAG) fractions were separated via solid-phase extraction according to Burdge et al [19]. Briefly, *75-100 mg of total lipid was suspended in chloroform and applied to amino-propyl silica columns (Supelclean TM LC-NH2 1 mL SPE columns, Supelco, Bellefonte, PA, USA) under gravity.…”

“…The column was washed with 2 mL of hexane followed by 2 mL of hexane:chloroform;ethyl acetate solution (100:5:5 v/v) to elute the CE and TAG fractions, respectively. Burdge et al [19] also allows for separation of free fatty acids; however, amounts recovered from fillet total lipid were extremely small (0.1-0.2 mg) and insufficient for subsequent FA analysis. Once separated, each lipid class was evaporated to dryness under nitrogen and weighed to determine total tissue PL, CE, and TAG content.…”

Preparation of Fatty Acid Methyl Esters by Selective Methanolysis of Polar Glycerolipids

“…After phase separation, the chloroform layer is washed with water, and concentrated to dryness. Total PLs are isolated as polar lipids from total lipids by thin-layer chromatography (TLC) or column chromatography [14][15][16]. Total PLs are scraped off the TLC plates or total PLs eluted through a column are concentrated to dryness.…”