AbstractsBiology & Animal Science

Neurotransmitters and neurosteroids are compounds that regulate the functions of the brain. The neurotransmitters dopamine (DA) and serotonin (5-HT) play a role in several psychological conditions, including schizophrenia, depression and anxiety. DA also has an important role in Parkinson s disease. Neurosteroids are involved in neurodegenerative diseases. In Alzheimer s disease and multiple sclerosis, the levels of neurosteroids are decreased in certain areas of the brain. Neurosteroids differ from classical neurotransmitters in that they are lipid-soluble and can easily cross the blood-brain barrier (BBB). Neurotransmission can be studied in vivo by microdialysis, and as the concentrations of neurotransmitters in the microdialysates are very low, sensitive analytical methods are needed for their analysis. In this work an UPLC-MS/MS method was developed for the determination of 5-HT, DA, their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfonate and glucuronide conjugates. The method was validated and applied for analyzing human brain microdialysis and cerebrospinal fluid (CSF) samples. Intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain. The origin of the determined phase II metabolites in the brain is unknown. Even though sulfonate-conjugated compounds such as dopamine sulfonate (DA-S) and 5-HIAA-S were detected in the human brain, it is unclear whether they were locally formed or transported into the brain through the BBB from peripheral sources. The BBB permeation of DA-S was studied by administration of isotope (13C6)-labelled DA-S, which can be distinguished from endogenous DA-S by mass spectrometry, subcutaneously (s.c.) while brain microdialysis samples were collected and analyzed by UPLC-MS/MS. The fate of 13DA-S in brain was followed by monitoring 13C6-labelled DA-S metabolites and hydrolysis products. The results proved that DA-S permeates through the BBB, and indicated that DA-S finally either permeates through the BBB back to the peripheral circulation or is dissociated or metabolized by unknown mechanisms. While the hydrophilic neurotransmitters DA and 5-HT are well suited for analysis by liquid chromatography coupled to atmospheric pressure ionization, the neurosteroids have more commonly been analyzed by methods based on gas chromatography (GC) coupled to ionization in vacuum. Recently GC has been combined to atmospheric pressure photoionization utilizing heated nebulizer microchips (μAPPI). We now constructed a simpler interface for combining GC to mass spectrometry (MS) using dopant-assisted atmospheric pressure photoionization (APPI), utilizing commercially available hardware. The neurosteroids were analyzed as trimethylsilyl (TMS) derivatives, and the effect of different dopants (chlorobenzene, toluene and anisole) on the ionization and on the sensitivity of the method was investigated. Chlorobenzene was chosen as the best dopant, as the neurosteroid-TMS derivatives formed intense molecular ions with minimal fragmentation,…