A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction
Gc. Burdge et al., A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction, BR J NUTR, 84(5), 2000, pp. 781-787
Efficient isolation of individual lipid classes is a critical step in the a
nalysis of plasma and lipoprotein fatty acid compositions. Whilst good sepa
rations of total lipid extracts are possible by TLC, this method is time co
nsuming and a major rate-limiting step when processing large numbers of spe
cimens. A method for rapid separation of phosphatidylcholine (PC), non-este
rified fatty acids (NEFA), cholesterol ester (CE) and triacylglycerol (TAG)
from total plasma lipid extracts by solid-phase extraction (SPE) using ami
nopropyl silica columns has been developed and validated. Following initial
separation of polar and neutral lipids, individual classes were isolated b
y 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 wer
e significantly greater for TAG and NEFA than by TLC (P<0.001). Both GC-fla
me ionisation detector and GC-MS analysis of fatty acid methyl esters demon
strated that there was no cross-contamination between lipid classes. Measur
ements 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 ap
peared 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 alterna
tive to TLC for isolation of these lipid classes from plasma.