Simultaneous Measurement of Serotonin, Dopamine and Their Metabolites in Mouse Brain Extracts by High-Performance Liquid Chromatography with Mass Spectrometry Following Derivatization with Ethyl Chloroformate

Abstract

In order to measure the levels of serotonin (5-hydroxyltryptamine, 5-HT), dopamine (DA), 3,4-hydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) simultaneously, an effective derivatization method followed by high-performance liquid chromatography (HPLC) coupled to electrochemical ionization mass spectrometry was used. The derivatization reaction of biological samples with ethyl chloroformate occurred rapidly at room temperature in aqueous conditions, and the resulting derivatives were isocratically separated with good selectivity using a C18 reversed-phase column within 30 min. The study results showed that the new derivatization procedure offers an excellent means of simultaneous determination of 5-HT, DA and their metabolites in mouse brain homogenates, which are important in a number of physiological and behavioral functions.Key words neurotransmitter; HPLC-MS; ethyl chloroformate; derivatization Dopamine (DA) and serotonin (5-hydroxyltryptamine, 5-HT), which are widely distributed as a monoamine neurotransmitter in the central nervous system (CNS), 1) play important physiological and behavioral roles such as the control and regulation of affective behavior, sleep, reward system, motor and cognitive functions.2) Changes of DA and 5-HT basal levels have been linked to some CNS-related disorders such as depression, panic disorder, schizophrenia and Parkinson's disease (PD).2,3) PD is pathologically characterized by the death of dopaminergic neurons in the substantia nigra pars compacta 4) and caused by neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahypyridine (MPTP), which produces a clinical syndrome similar to PD. Because the administration of MPTP is known to affect the dopaminergic neurons in the nigrostriatal system with subsequent depletion of DA and its metabolites, 5,6) the measurement of 5-HT, DA and their metabolites is necessary to discover and test new drugs for treating these disorders. 7)Various analytical methods have been widely investigated for determining the level of monoamine neurotransmitters and their metabolites, including high-performance liquid chromatography (HPLC) coupled with electrochemical detection (ECD), dual ECD, fluorescence detection (FLD), FLD/ECD system, chemiluminescence detection and mass spectrometer (MS) and gas chromatography (GC) coupled with MS in biological samples such as plasma, rat brain, and mouse brain and in vitro experiments.8-10) HPLC-ECD, which is the most common analytical technique used to determine monoamines and their metabolites, has the advantages of low cost and fast speed 11) but maintaining its routine use is problematic due to sudden noise problems.12) The HPLC-FLD method is used to measure fluorescent derivatives of monoamine neurotransmitters by a derivatization procedure, 13) which was performed by benzylamine and 1,2-diphenylethylenediamine in a two-step reaction in weakly alkaline media and in the presence of potassium hexacyanoferrate (III).14) However, since this protocol requires the vigorous reaction ...

Distribution, properties, and inhibitor sensitivity of zebrafish catechol-O-methyl transferases (COMT)

“…The contribution of COMT and MAO to dopamine inactivation can be inferred from the levels of dopamine and its metabolites (3-MT, the product of methylation, and DOPAC, the oxidation product) in the normal tissue. The levels of both DOPAC and 3-MT in the mouse striatum [66] and in the entire right hemisphere [67] were lower than the levels of dopamine, and the levels of 3-MT were lower than the levels of DOPAC. The levels of 3-MT reportedly were the same or higher than the levels of DOPAC in most part of the chick brain (except the optic lobes and the cerebellum) [68], and in all regions of the trout brain [69].…”

Olfactory bulb monoamine concentrations vary with time of day

“…Thus, we used DOPAC as our measure of DA activity. However, other groups report higher concentrations of HVA than DOPAC in some brain regions (Gomez et al, 2007; Park et al, 2013; Zant et al, 2011), and one report shows higher HVA than DOPAC in specific brain regions but higher DOPAC than HVA overall (Laatikainen et al, 2013). While it is possible that re-analysis of our data using HVA would alter the amplitude of the observed DA/metabolite ratio fluctuations, our interpretations of the results would be unlikely to change.…”

Analysis and measurement of serotonin

“…For example, single quadrupole LC/MS was utilized for the analysis of serotonin and melatonin in plants (Erland, Chattopadhyay, Jones, & Saxena, ). Atmospheric pressure ionization techniques such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have been used in LC/MS to analyze serotonin and its metabolites in a variety of samples (Carrera, Sabater, Vilanova, & Sogorb, ; Park et al, ). As an example, LC/MS with ESI was used to quantify serotonin and its metabolite, 3‐methoxytyramine, in mouse brain extracts with sensitivity in the low ng/mL range, but the method required derivatization (Park et al, ).…”

“…LC/MS/MS systems can use ESI (Santos‐Fandila, ) or APCI (Zhou et al, ) techniques as is the case with LC/MS instruments (Carrera et al, ; Park et al, ). For example, tryptophan and eight metabolites, including serotonin, were analyzed in human plasma with an ESI LC/MS/MS method that was validated according to ISO 15189 (Boulet, Faure, Flore, Montérémal, & Ducros, ), and an APCI LC/MS/MS method was used to quantify serotonin, 5‐HIAA and tryptophan in human whole blood followed by the cross‐validation of the results with a HPLC/FL method from an external laboratory.…”

“…C/AEx, cation/anion exchange column; CE/MS, capillary electrophoresis with mass spectrometry; DP, deproteinization; EI, electron impact ionization; ESI, electrospray ionization; GC/ECD, gas chromatography with electron capture detection; GC/FID, gas chromatography with flame ionization detection; HPLC/AMP, high-performance liquid chromatography with amperometric detection; HPLC/FL, high-performance liquid chromatography with fluorometric detection; LC/MS 3 , liquid chromatography with tandem mass spectrometry cubed; LE, liquid extraction; MEL, melatonin; NAS, N-acetylserotonin; NCI, negative chemical ionization; nLC/MS/MS, nanoflow liquid chromatography with triple quadrupole mass spectrometry; NT, neurotransmitter; SPE, solid-phase extraction; TRP, tryptophan. (Carrera et al, 2007;Park et al, 2013). For example, tryptophan and eight metabolites, including serotonin, were analyzed in human plasma with an ESI LC/MS/MS method that was validated according to ISO 15189 (Boulet, Faure, Flore, Montérémal, & Ducros, 2017), and an APCI LC/MS/MS method was used to quantify serotonin, 5-HIAA and tryptophan in human whole blood followed by the cross-validation of the results with a HPLC/FL method from an external laboratory.…”