Validation of a Reversed Phase UPLC-MS/MS Method to Determine Dopamine Metabolites and Oxidation Intermediates in Neuronal Differentiated SH-SY5Y Cells and Brain Tissue

Abstract

Dopamine is a key neurotransmitter in the pathophysiology of various neurological disorders such as addiction or Parkinson’s disease. Disturbances in its metabolism could lead to dopamine accumulation in the cytoplasm and an increased production of o-quinones and their derivatives, which have neurotoxic potential and act as precursors in neuromelanin synthesis. Thus, quantification of the dopaminergic metabolism is essential for monitoring changes that may contribute to disease development. Here, we developed and validated an UPLC-MS/MS method to detect and quantify a panel of eight dopaminergic metabolites, including the oxidation product aminochrome. Our method was validated in differentiated SH-SY5Y cells and mouse brain tissue and was then employed in brain samples from humans and rats to ensure method reliability in different matrices. Finally, to prove the biological relevance of our method, we determined metabolic changes in an in vitro cellular model of dopamine oxidation/neuromelanin production and in human postmortem samples from Parkinson’s disease patients. The current study provides a validated method to simultaneously monitor possible alterations in dopamine degradation and o-quinone production pathways that can be applied to in vitro and in vivo experimental models of neurological disorders and human brain samples.

Brain Microdialysis Coupled to LC-MS/MS Revealed That CVT-10216, a Selective Inhibitor of Aldehyde Dehydrogenase 2, Alters the Neurochemical and Behavioral Effects of Methamphetamine

“…DA, 5-HT, and their metabolites are low-molecular-weight polar analytes; thus, separation and detection using the LC-MS system are not easy tasks. According to previous studies, sample introduction or preparation techniques such as direct injection, solid phase extraction, online enrichment, and chemical derivatization using benzoyl chloride (BzCl) and dansyl chloride were used to overcome low sensitivity. ,− Most neurotransmitters retain weakly on reversed-phase columns because of their high polarity. In this study, DA, 5-HT, and their metabolites obtained in rat brain microdialysates were analyzed by derivatization into relatively nonpolar analytes by benzoylation of the primary and secondary amine groups.…”

In vivoreduction of age-dependent neuromelanin accumulation mitigates features of Parkinson’s disease

“…UPLC-MS/MS analysis of rat brain regions was performed using our previously validated method 20 with some modifications. Each sample was injected three times into the UPLC-MS/MS system to analyse different sets of compounds i.e.…”

“…Aminochrome standard (0.5 mM) was freshly prepared as previously described. 20 , 21 Multiple Reaction Monitoring (MRM) acquisition settings for the targeted metabolites are summarized in Table 2 . Samples with a concentration between LOD and LOQ or bigger than LOQ were considered acceptable; samples with a concentration lower than LOD were considered as the LOD value.…”

In vivo reduction of age-dependent neuromelanin accumulation mitigates features of Parkinson’s disease

“…UPLC-MS/MS analysis of rat brain regions was performed using our previously validated method 19 with some modifications. Each sample was injected three times into the UPLC-MS/MS system to analyze different sets of compounds i.e.…”

“…5SCD and 5SCDA standards were kindly donated by Professor Kazumasa Wakamatsu and Professor Shosuke Ito at the Fujita Health University, Aichi, Japan. Aminochrome standard (0.5 mM) was freshly prepared as previously described 19,20 . Multiple Reaction Monitoring (MRM) acquisition settings for the targeted metabolites are summarized in Table 2.…”