Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry

A mass spectrometric method for the quantification of free cholesterol in c ells and subcellular membranes is presented. The method is based on a simpl e one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano- electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a st able isotope labeled internal standard. The determination of free cholester ol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell Line, With this method a molar ratio of free cholesterol to total phosphol ipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membran e fraction enriched in Golgi membranes, a molar ratio of free cholesterol t o total phospholipids of 0.57 mol/mol was determined, The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol.