Highly sensitive isotope-dilution liquid-chromatography–electrospray ionization–tandem-mass spectrometry approach to study the drug-mediated modulation of dopamine and serotonin levels in Caenorhabditis elegans

Abstract

Dopamine (DA) and serotonin (SRT) are monoamine neurotransmitters that play a key role in regulating the central and peripheral nervous system. Their impaired metabolism has been implicated in several neurological disorders, such as Parkinson’s disease and depression. Consequently, it is imperative to monitor changes in levels of these low-abundant neurotransmitters and their role in mediating disease. For the first time, a rapid, specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of DA and SRT in the nematode Caenorhabditis elegans (C. elegans). This model organism offers a unique approach for studying the effect of various drugs and environmental conditions on neurotransmitter levels, given by the conserved DA and SRT biology, including synaptic release, trafficking and formation. We introduce a novel sample preparation protocol incorporating the usage of sodium thiosulfate in perchloric acid as extraction medium that assures high recovery of the relatively unstable neurotransmitters monitored. Moreover, the use of both deuterated internal standards and the multiple reaction monitoring (MRM) technique allows for unequivocal quantification. Thereby, to the best of our knowledge, we achieve a detection sensitivity that clearly exceeds those of published DA and SRT quantification methods in various matrices. We are the first to show that exposure of C. elegans to the monoamine oxidase B (MAO-B) inhibitor selegiline or the catechol-O-methyltransferase (COMT) inhibitor tolcapone, in order to block DA and SRT degradation, resulted in accumulation of the respective neurotransmitter. Assessment of a behavioral output of the dopaminergic system (basal slowing response) corroborated the analytical LC-MS/MS data. Thus, utilization of the C. elegans model system in conjunction with our analytical method is well-suited to investigate drug-mediated modulation of the DA and SRT system in order to identify compounds with neuroprotective or regenerative properties.

Regulation and modulation of biogenic amine neurotransmission in Drosophila and Caenorhabditis elegans

“…When wildtype worms were exposed to a MAO-B inhibitor, DA levels were increased. The same results were seen when COMT inhibitor was applied ( Schumacher et al, 2015 ). Both studies suggest that an MAO-like mechanism exists for the degradation of DA.…”

A Reliable Method Based on Liquid Chromatography–Tandem Mass Spectrometry for the Simultaneous Quantification of Neurotransmitters in Caenorhabditis elegans

“…Despite the usage of behavioral assays and the microscopy of fluorescence-tagged neurons, only a few chromatographic approaches have been carried out in C. elegans to quantify DA. Only Schumacher et al displayed a validated LC-MS/MS-based method to analyze DA and SRT in C. elegans , but they excluded GABA and ACh [ 30 ], which are, however, essential for the investigation of neurotoxicity [ 10 , 58 ]. Using our method, we were able to determine neurotransmitter profiles in wildtype worms, as well as in cat-2Δ and ace-1Δ::ace-2Δ worms.…”

“…Other assays, namely those of locomotion, are mediated by several neurotransmitters, such as acetylcholine and dopamine; these provide broader outcomes, but can be problematic in interpretation. So far, only Schumacher et al have assessed DA and SRT [ 30 ], but, to date, GABA and ACh have not been quantified in C. elegans . Therefore, a method is required for the simultaneous quantification of multiple neurotransmitters in C. elegans .…”

Simultaneous detection of dopamine, tyrosine and ascorbic acid using NiO/graphene modified graphite electrode

“…Though analytical methods like chro matography, fluorescence, mass spectrometry and other techniques are available for the detection of Tyr [19][20][21][22], DA [23][24][25][26][27][28][29][30][31] and AA [32][33][34][35][36][37][38] in biological fluids, they are useful to certain extent due to their own limitations. Owing to fast response, accuracy and portability, the electrochemical methods using electrochemical sensors [39] to analy ze the DA, AA and Tyr coexisting in biological flu id [40][41][42][43] are very promising in accurate quantification of these bio-molecu les.…”