RAPID SEPARATION AND QUANTITATION OF COMBINED NEUTRAL AND POLAR LIPIDCLASSES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND EVAPORATIVE LIGHT-SCATTERING MASS DETECTION

Modifications are described for an innovative and widely used high-per formance liquid chromatography technique that resolves a very broad sp ectrum of lipids for quantitation by evaporative light-scattering dete ction. Substitution of acetone for 2-propanol in a portion of the solv ent gradient program yields consistent resolution of diacylglycerol an d cholesterol without sacrificing baseline resolution of the remaining major lipid classes. Moreover, previously noted instabilities in tria cylglycerol retention time are eliminated. The introduction of acetone also enables a 20% reduction in flow-rate without an increase in tota l run time. As a further modification of the mobile phase composition, acetic acid and ethanolamine are substituted for the serine-ethylamin e combination that was originally shown to improve column performance. The combination of acetic acid and ethanolamine yields the same resul t but the increased volatility of these solutes over serine results in decreased baseline noise. Finally, 1,2-hexadecanediol is introduced a s an internal standard that is well suited for this method. The chroma tographic performance obtained with these modifications is demonstrate d in compositional analyses of lipid extracts from rat liver, heart, k idney and brain. (C) 1998 Elsevier Science B.V.