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

Abstract

In this study, a simultaneous assay for catecholamines and their metabolites in the brain was established using liquid chromatography-mass spectrometry (LC-MS). To achieve complete separation, a cation-exchange/reversed-phase mixedmode copolymer resin column containing 0.81 wt% sulfo groups was used for the simultaneous LC-MS assay. The analyzed catecholamines were dopamine (DA), norepinephrine (NE), and epinephrine (E), while the metabolites lacking amino groups were 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxy-4hydroxyphenylglycol (MHPG). The metabolites were separated and detected using LC-MS, on columns with and without sulfo groups. However, we could not achieve adequate separation of catecholamines on both columns using a gradient elution of 0 -50 (v/v)% methanol containing 0.1 (v/v)% formic acid (FA). When volatile ion-pairing reagents were added to the mobile phase, they improved the retention and detection of catecholamines on the sulfonated mixed-mode column. Under optimized elution conditions, which involved a linear gradient elution of water containing 0.1 (v/v)% FA to 50 (v/v)% acetonitrile in 50 mM ammonium formate at 40 C and a 0.20 mL/min rate, all six target molecules were simultaneously detected within 25 min, when using negative mode LC-MS on a sulfonated mixed-mode column. The limits of detection (LODs) for DA, NE, E, DOPCA, HVA, and MHPG were determined to be 20. 7, 12.6, 74.6, 1110, 18.7, and 3196 nM, respectively. Moreover, the established LC-MS assay allowed the detection of endogenous DA, NE, and HVA, in normal mouse brain samples at concentrations higher than 20, 9, and 4 pmol/mg, respectively.

Biosensing Dopamine and L-Epinephrine with Laccase (Trametes pubescens) Immobilized on a Gold Modified Electrode

“…Despite the importance of NTs for clinical diagnosis, the existing quantification methods still need improvements in terms of selectivity [ 22 ], sensitivity, flexibility and simplification [ 23 ]. In an attempt to overcome the limitations inherent to the routinely used diagnostic kits for the determination of catecholamines, chromatography [ 24 , 25 ], chemiluminescence [ 26 ], electrophoresis [ 27 ] or electroanalysis with chemically modified electrodes [ 28 , 29 , 30 , 31 ] have been proposed.…”

A chromatographic approach for studying the adsorption of polar small molecules on tetrabutylammonium bromide semiclathrate hydrate

“…Liquid chromatography (LC), while it has been widely used for analytical separation, [20][21][22] can also be a powerful tool for understanding the interaction mechanism between the solutes and stationary phases with varying experimental conditions such as temperature and the composition of liquid phase. [22][23][24] Tasaki and Okada [25][26][27][28][29][30] proposed a novel LC system using a column packed with water-ice particles and found that the retention mechanism of small molecules on the surface of ice changes with temperature and the mobile phase composition, which was caused by the structural change in the water-ice surface.…”

Efficient Sub-1 Minute Analysis of Selected Biomarker Catecholamines by Core-Shell Hydrophilic Interaction Liquid Chromatography (HILIC) with Nanomolar Detection at a Boron-Doped Diamond (BDD) Electrode

“…Various analytical techniques for CAs analysis have been reported. Reverse-phase liquid chromatography (RPLC) using C18 columns hyphenated to different detection systems, including mass spectrometry (MS) [21][22][23], electrochemical detection (ECD) [24][25][26][27][28], and fluorescence detection [29] has been widely applied. However, due to the non-polar character of C18 phases and the high polarity of the CAs, derivatization steps, as well as ion pair additives are often considered.…”