Highly sensitive assay for the measurement of serotonin in microdialysates using capillary high-performance liquid chromatography with electrochemical detection

A rapid and sensitive method for the analysis of brain monoamine neurotransmitters using ultra-fast liquid chromatography coupled to electrochemical detection

“…For the Kinetex 2.6 m column, we optimized the temperature of the column oven at 25 • C in order to separate the closest peaks in the run, these being 3-MT and 5-HT (Rs = 0.95, Rs values between other peaks were greater than 1.5), and chose 35 • C for detection, as recommended by the supplier of the EC cell [6]. The final conditions were: mobile phase: 0.07 mol/L potassium phosphate, 0.1 mmol/L EDTA, 1.1 mmol/L OSA, 3.1 mmol/L triethylamine, 14% methanol, pH 3.12, elution at 1.6 mL/min at 25 • C. The order of elution was the same as with the conventional HPLC column, namely NA < DHBA < DOPAC < DA < 5-HIAA < HVA < 3-MT < 5-HT ( Fig.…”

“…The use of a smaller diameter (2 mm) column and 1.7 m particles offered only a limited improvement, as a 5 L volume sample was needed to obtain a suitable LOD for dialysate analyses (data not shown). In order to analyze neurotransmitters in lower sample volumes, capillary columns with diameters less than 1 mm are required, as previously reported by our group (detection of 0.084 fmol of 5-HT in a 1 L sample using a 150 mm × 0.5 mm C18 capillary column with 5 m particles) [6].…”

“…The main advantage of coulometry is the full oxidation of the compounds entering the detection cell, resulting in a low limit of detection (LOD); however, the miniaturization of the cell is currently limited. In contrast, the size of the amperometric cell can be easily reduced and, when coupled to micro-bore and capillary columns [6], amperometry can detect low levels of compounds despite a weak electrochemical yield (∼10%).…”

Reassessment of the antioxidative mixture for the challenging electrochemical determination of dopamine, noradrenaline and serotonin in microdialysis samples

“…5-HT is found stable in Ringer's solution at pH 6 yet unstable in acidified solutions [23][24][25][26]. Furthermore, instability of 5-HT in artificial cerebrospinal fluid but stability in microdialysis samples has been reported [27]. Nonetheless, considering simultaneous monoamine analysis, stability of both catecholamines and indolamines is required for samples kept in the autosampler prior to analysis as well as during long term storage, usually under freezing conditions.…”

A Simple High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Monoamine Neurotransmitters and Relative Metabolites with Application in Mouse Brain Tissue

“…[15] There are several reported methods utilizing capillary electrophoresis with ECD for such determinations, but they also suffer from extended analytical run time (∼40 min) or inability to quantitate all of the components simultaneously, as achieved by our currently developed method. [16,17] A recent method utilized ECD to determine the concentration of catecholamines, kynurenine, and indole derivatives of tryptophan in rat brain tissue homogenate with a simple sample preparation, but this method also required an extensive analytical run time (∼20 min) and the use of high electrode potentials for some of the analytes, which can be damaging to the electrochemical cells over time. [18] One method describing analysis of the kynurenine metabolites from the tryptophan pathway (i.e., quinoloinic acid, nicotinic acid, and xanthurenic acid) was conducted with the use of simultaneous ultraviolet and fluorimetric detection requiring a derivitization step, as compared to the proposed simple sample preparation of the current method, and was only described in aqueous clean solutions.…”