A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid

Rohini Sidhu,Hui Jiang,Nicole Y Farhat,Nuria Carrillo-Carrasco,Myra Woolery,Elizabeth Ottinger,Forbes D Porter,Jean E Schaffer,Daniel S Ory,Xuntian Jiang

doi:10.1194/jlr.d058487

Abstract

24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker.

Highlights

24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain
24(S)-HC in plasma can be detected by ESI as [M+NH4]+ on a 4000QTRAP and by atmospheric pressure chemical ionization (APCI) as [M+H-H2O]+ on Thermo TSQ triple-quadrupole mass spectrometers, the sensitivities are insufficient to detect unesterified 24(S)-HC in cerebrospinal fluid (CSF)
The intended use of the assay was for measuring the 24(S)-HC as a pharmacodynamic biomarker in clinical trials, and the data generated to be used for critical decision-making in the development of HP-␤-CD as new drug candidate

Summary

Introduction

24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. A validated LCMS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of increased cholesterol metabolism in the CNS. LC-MS/MS was demonstrated as a sensitive, specific, and rapid method for the quantification of 24(S)-HC in biological samples. While highly abundant 24(S)-HC in plasma and serum [23, 25, 27, 28, 30, 35] has been analyzed by LC-MS/MS and GC-MS [20,21,22], measurement of low abundant 24(S)-HC in CSF by GC-MS requires large sample volumes [37]

Methods

Results

Conclusion