抄録
Analysis of neurotransmitters is a critical tool in the evaluation of functional neurotoxicity, and when localized to particular brain regions, mechanisms of toxicity can be more precisely elucidated. This laboratory validated a method for determination of dopamine (DA), serotonin (5HT) and 2 DA metabolites, DOPAC and HVA, using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in the positive (amines) and negative (acids) electrospray ionization mode. DA, 5HT, DOPAC and HVA were quantitated in a single 5-minute run at respective LLOQs of 10, 10, 30 and 30 pg/mg wet wt using up to 200 µL of a 10% (w/v) tissue homogenate sample. C57BL/6J mouse striata were collected at 1 mm thicknesses using brain molds and snap frozen in liquid N2 within 5-6 minutes to minimize artefactual changes in endogenous neurotransmitter levels. Frozen striata were homogenized in diluent containing deuterated DA, 5HT, DOPAC and HVA as internal standards. Homogenates were extracted using Polymeric RP SPE cartridges and the extracts analyzed by LC-MS/MS. The following means ± SD (expressed as ng/mg wet wt) of 9-10 striata were obtained for DA – 9.29 ± 2.12, 5HT – 0.387 ± 0.072, DOPAC – 1.63 ± 0.45 and HVA – 1.32 ± 0.20. The means were consistent with normally expected ranges in the literature and confirm that LC-MS/MS is a sensitive method for quantitation of these analytes extracted from striatal tissues.
