A simple and sensitive high‐performance liquid chromatography–electrochemical detection assay for the quantitative determination of monoamines and respective metabolites in six discrete brain regions of mice

Abstract

A rapid, sensitive, and reproducible assay is described for the quantitative determination of the monoamine neurotransmitters dopamine, norepinephrine and serotonin, their metabolites, and the internal standard 3,4-dihydroxybenzlyamine hydro-bromide in mouse brain homogenate using high-performance liquid chromatography with electrochemical detection. The method was validated in the following brain areas: frontal cortex, striatum, nucleus accumbens, hippocampus, substantia nigra pars compacta and ventral tegmental area. Biogenic amines and relevant metabolites were extracted from discrete brain regions using a simple protein precipitation procedure, and the chromatography was achieved using a C column. The method was accurate over the linear range of 0.300-30 ng/mL (r = 0.999) for dopamine and 0.300-15 ng/mL (r = 0.999) for norepinephrine, 3,4-dihydroxybenzlyamine hydro-bromide, homovanillic acid and 5-hydroxyindolacetic acid, with detection limits of ~0.125 ng/mL (5 pg on column) for each of these analytes. Accuracy and linearity for serotonin were observed throughout the concentration range of 0.625-30 ng/mL (r = 0.998) with an analytical detection limit of ~0.300 ng/mL (12 pg on column). Relative recoveries for all analytes were approximately ≥90% and the analytical run time was <10 min. The described method utilized minimal sample preparation procedures and was optimized to provide the sensitivity limits required for simultaneous monoamine and metabolite analysis in small, discrete brain tissue samples.

Comprehensive analysis of nine monoamines and metabolites in small amounts of peripheral murine (C57Bl/6 J) tissues

“…Deviations in biological samples were <15% for all analytes and generally <10%, as suggested by the US Food and Drug Administration's requirements for bioanalytical method validation (Table ). These values are also in accordance with Zhang et al () and Allen et al (). The only exception to this is MHPG.…”

“…The LOD ranged from 0.625 to 2.5 pg/μL, while the LOQ was between 2.5 and 5 pg/μL (Table ). These values are in the same range or lower than those Zhang et al () achieved with a boron‐doped electrode, but four to five fold higher than the ones found by Allen et al ().…”

“…This shortened the run time by more than half from 40 min with the previous method (Supporting Information, 3) to 13 min with the current method. This is between the times of two recent methods developed by Allen et al (2017) and Zhang et al (2016). Figure 2 shows a chromatogram of a standard solution (Retention times shown in Supporting Information, 1).…”

“…ECD is also much more specific for the monoamines than MS detectors. Therefore, although in recent decades several other detectors have been tested for the quantification of such derivatives, high-performance liquid chromatography (HPLC) with ECD is still considered one of the best choices, indicated by the high number of reports (Allen, Rednour, Shepard, & Pond, 2017;Bidel et al, 2016;Birbeck & Mathews, 2013;Ferry, Gifu, Sandu, Denoroy, & Parrot, 2014;Ganesana, Lee, Wang, & Venton, 2017;Nguyen, Aerts, Van Dam, & De Deyn, 2010;Parrot, Neuzeret, & Denoroy, 2011;Schou-Pedersen, Hansen, Tveden-Nyborg, & Lykkesfeldt, 2016;Smith, Schwartz, & Lucot, 2013;Tsunoda, 2006).…”

Analysis of Catecholamine and Their Metabolites in Mice Brain by Liquid Chromatography-Mass Spectrometry Using Sulfonated Mixed-mode Copolymer Column

“…This indicated that the urethane-anesthetized mice used in this study might not be exposed to excess nervous stress or promotion of the brain nervous system. 21,22 The amounts of DA, NE, and HVA detected in the brain samples from the three mice using the aforementioned LC-TOF/MS procedures ranged from 21.6 to 29.0, 9.0 to 9.2, and 4.3 to 5.7 pmol/mg, respectively ( Table 2), and were comparable to the reported levels of DA, NE, and HVA (in pmol) in brain tissue samples. 22 As shown in Fig.…”