Modifications are described for an innovative and widely used high-performance liquid chromatography technique that resolves a very broad spectrum of lipids for quantitation by evaporative light-scattering detection. Substitution of acetone for 2-propanol in a portion of the solvent gradient program yields consistent resolution of diacylglycerol and cholesterol without sacrificing baseline resolution of the remaining major lipid classes. Moreover, previously noted instabilities in triacylglycerol retention time are eliminated. The introduction of acetone also enables a 20% reduction in flow-rate without an increase in total run time. As a further modification of the mobile phase composition, acetic acid and ethanolamine are substituted for the serine-ethylamine combination that was originally shown to improve column performance. The combination of acetic acid and ethanolamine yields the same result but the increased volatility of these solutes over serine results in decreased baseline noise. Finally, 1,2-hexadecanediol is introduced as an internal standard that is well suited for this method. The chromatographic performance obtained with these modifications is demonstrated in compositional analyses of lipid extracts from rat liver, heart, kidney and brain.