A fast method to quantify free and esterified cholesterol in human plasma and disease models

Cholesterol exists in two forms in the human body: free and esterified as cholesteryl esters (CE). Free cholesterol is an essential component of cell membranes and participates in the biosynthesis of bile acids and steroid hormones. Cholesterol is transported as CE in lipoprotein particles and stored in cells as part of lipid droplets. Here, we present a rapid method (4 min) capable of analyzing both cholesterol forms (free and esterified) in a single experiment, utilizing supercritical fluid chromatography (SFC) coupled with high-resolution mass spectrometry (QTOF). This method takes advantage of the commonly observed, yet often undesired, in-source fragmentation of cholesteryl esters during electrospray ionization (ESI). In addition, it offers the advantage of measuring the abundance of molecular CE species, and it is suitable for low-volume samples as it can effectively analyze plasma or serum diluted up to 100-fold, requiring only a simple protein precipitation step for sample preparation. The method was validated using human plasma reference materials, showing excellent accuracy as demonstrated by the analysis of the NIST Standard Reference Material (SRM) 1950. Furthermore, the performance of this workflow for biomedical studies was tested in a large human Asian cohort for Coronary Heart Disease and in a cellular model of Niemann-Pick disease type C1 (NPC1), to follow separate pathways of cellular cholesterol metabolism.