Determination of seven selected neuro- and immunomodulatory steroids in human cerebrospinal fluid and plasma using LC-MS/MS

Abstract

Dehydroepiandrosterone (DHEA) and its 7-oxo- and 7-hydroxy-metabolites occurring in the brain are considered neurosteroids. Metabolism of the latter is catalysed by 11β-hydroxysteroid dehydrogenase (11β-HSD) which also interconverts cortisol and cortisone. The concurrent metabolic reaction to DHEA 7-hydroxylation is the formation of 16α-hydroxy-DHEA. The LC–MS/MS method using triple stage quadrupole-mass spectrometer was developed for simultaneous quantification of free DHEA, 7α-hydroxy-DHEA, 7β-hydroxy-DHEA, 7-oxo-DHEA, 16α-hydroxy-DHEA, cortisol and cortisone in human plasma and cerebrospinal fluid (CSF). The method employs 500μL of human plasma and 3000μL of CSF extracted with diethyl ether and derivatized with 2-hydrazinopyridine. It has been validated in terms of sensitivity, precision and recovery. In plasma, the following values were obtained: limit of detection: 2–50pg/mL; limit of quantification: 5–140pg/mL; within-day precision 0.58–14.58%; between-day precision: 1.24–13.89% and recovery: 85–113.2%). For CSF, the values of limit of detection: 2–28pg/mL; limit of quantification: 6–94pg/mL; within-day precision; 0.63–5.48%; between-day precision: 0.88–14.59% and recovery: 85.1–109.4% were acquired. Medians and concentration ranges of detected steroids in plasma and CSF are given in subjects with excluded normal pressure hydrocephalus (n=37; 65–80years). The method enables simultaneous quantification of steroids important for the estimation of 11β-HSD activity in human plasma and CSF. It will be helpful in better understanding various degenerative diseases development and progression.