Liquid chromatography-tandem mass spectrometry determination of plasma 24S-hydroxycholesterol with chromatographic separation of 25-hydroxycholesterol

Abstract

Concentrations of circulating 24S-hydroxycholesterol (24SOHChol) are of interest as a practical measure of cholesterol efflux from the human brain. The current method of choice for 24SOHChol quantification is with gas chromatography-mass spectrometry (GC-MS). Liquid chromatography-mass spectrometry (LC-MS) methods to detect 24SOHChol have been described, but they lack rigorous high-performance liquid chromatography (HPLC) separation of a closely eluting isomeric oxysterol, 25-hydroxycholesterol. This is important because 25-hydroxycholesterol can be present in significant amounts and tandem mass spectrometry (MS/MS) cannot completely differentiate 24SOHChol. We describe an LC-MS method with rapid chromatographic separation of the oxysterols to permit accurate determination of plasma 24SOHChol. The availability of an LC-MS method offers advantages such as simplified sample work-up and analysis.

Figure 1

Both 24OHChol and 25-hydroxycholesterol are detected with MS/MS. To ensure HPLC separation of the oxysterols we initially used an isocratic methanol:acetonitrile:water mobile phase (45:40:35) modified from Burkard et al. [8] with an HPLC column temperature of 55°C. Oxysterol retention times were around 25 min with the 24R- and 24S-OHChol isomers separated from one another (Panel A authentic standards). An alternative eluent of methanol:acetonitrile:water (14:0.6:1) with a column temperature of 10°C enabled separation of 24OHChol from 25-hydroxycholesterol within 6.5 min (Panel B). Also shown are extracted ion chromatograms from plasma samples where significant 25-hydroxycholesterol was detected (Panels C and D).

Figure 2

Panel A is a Bland-Altman plot for the comparison of 24SOHChol concentration measured by GC/MS and LC/MS. The mean difference of −3.8ng/mL is indicated by the solid line, the 95% confidence intervals by dashed lines. Panel B is a Passing-Bablok regression analysis.