Determination of 24S- and 27-hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry

24S-Hydroxycholesterol (24S-OH-Chol) and 27-hydroxycholesterol (27-OH-Chol) are oxidized derivatives of cholesterol and of potential diagnostic interest because their circulating levels may reflect the cholesterol metabolism of the brain and macrophages, respectively. We developed a sensitive and specific HPLC-MS method for the quantification of 24S-OH-Chol and 27-OH-Chol in human plasma. In contrast to currently available procedures based on gas chromatography-mass spectrometry, this methodology offers the advantage that no time-consuming derivatization is needed. After saponification, solid-phase extraction, and HPLC separation under reversed-phase column conditions, detection by MS was performed using atmospheric pressure chemical ionization and selected ion monitoring mode. The standard curves were linear throughout the calibration range for both oxysterols. Within-day and between-day coefficients of variation were less than 9%, and the recoveries ranged between 98% and 103%. The quantification limits were 40 and 25 microg/l for 24S-OH-Chol and 27-OH-Chol, respectively. Mean values for both oxysterols were determined in plasma from 22 healthy volunteers. The sensitive and selective HPLC-MS method described here combined with the appropriate workup procedure allow the quantification of 24S-OH-Chol and 27-OH-Chol in plasma samples, for example in clinical studies to elaborate the clinical usefulness of these two oxysterols.