Development of an LC-MS/MS method for neurosteroids and its’ application in a stress-induced rat model

Abstract

A highly sensitive and rapid method based on liquid chromatography-quadrupole mass spectrometry (LC-MS/MS) has developed and validated for the simultaneous measurement of 17 neuroactive steroids (i.e. progesterone (PROG), 17-hydroxyprogesterone (17OH-PROG), 5α-dihydroprogesterone (5α-DHP), 5β-dihydroprogesterone (5β-DHP), estrone, androstenedione, cortisol, cortisone, corticosterone, dehydroepiandrosterone (DHEA), pregnanolone, allopregnanolone (AP), 11-deoxycorticosterone (DOC), 11-deoxycortisol, pregnenolone (PREG), 5α-tetrahydrodeoxycorticosterone (5αTHDOC), 5β-tetrahydrodeoxycorticosterone (5β-THDOC)) in rat brain and plasma. The brain and plasma neuroactive steroids were extracted with methanol:acetic acid (99:1). In the present method, neurosteroids were derivatized with a hydroxylamine reagent. All samples were processed using solid-phase extraction (SPE) with Oasis HLB cartridges. The separation of 17 neuroactive steroids was achieved within 13 min at a flow 0.4 mL/min with a C18 column (3.0×50 mm, 2.7 μm). The quadrupole mass spectrometer was operated in positive electro spray ionization mode. Each calibration curve showed good linearity (r2 >0.99). The intra-day and inter-day-assay coefficients of variation (CV) were less than 14.50% and 14.47%. The accuracy values of equivalent samples were less than 14.05% and 10.82%, respectively. Each stability test showed exellent stability. (<14.47%). The developed method was successfully applied to the simultaneous analysis of neuroactive steroids levels in a stress-induced rat model. These analytical tools may potentially help for the diagnosis of various neuronal disorders.