Integration of stable isotope labeling derivatization and magnetic dispersive solid phase extraction for measurement of neurosteroids by in vivo microdialysis and UHPLC-MS/MS.

In this work, a novel strategy of stable isotope labeling derivatization (SILD) combined with magnetic dispersive solid-phase extraction (MDSPE), has been proposed for simultaneous monitoring of neurosteroids changes linked to Parkinson's disease (PD) by in vivo microdialysis. The developed method was based on ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection using multiple-reaction monitoring (MRM) mode. In this study, a new pair of stable isotope labeling reagents d-/d-3-N-methyl-2'-carboxyl Rhodamine 6G (d-/d-MCR6G), were designed and synthesized for derivatizing neurosteroids in rat blood microdialysates and neurosteroid standards, respectively. d-MCR6G labeled neurosteroids standards were used as internal standard for the following pretreatment and UHPLC-MS/MS quantification to minimize the deviations caused by complex matrix and ion suppression effects in mass spectrometry analysis. Under the optimized derivatization and extraction conditions, good linearities of eight neurosteroids were obtained with correlation coefficients R values > 0.98. The limits of detection (LODs) and quantitation (LOQs) ranged from 0.06 to 0.12 pg/mL and 0.30-0.40 pg/mL, respectively. Taken together, the established method exhibited high sensitivity and selectivity, excellent accuracy, convenience and high efficiency. It was applied for the simultaneous and dynamic measurement of multiple neurosteroids in normal and PD rat blood microdialysates. This method would be expected to be potentially useful for the monitoring and drug treatment of PD and related neurological disorders in the future.