Optimization of ultra-performance liquid chromatography (UPLC) with fluorescence detector (FLD) method for the quantitative determination of selected neurotransmitters in rat brain

摘要

Background: Glutamate (Glu) and γ-aminobutyric acid (GABA) are the main neurotransmitters in the central nervous systemfor excitatory and inhibitory processes, respectively. Monitoring these neurotransmitters is an essential tool in establishingpathological functions, among others in terms of occupational exposure to toxic substances. Material and Methods: We presentmodification of the HPLC (high-performance liquid chromatography) to the UPLC (ultra-performance liquid chromatography)method for the simultaneous determination of glutamate and γ-aminobutyric acid in a single injection. The isocratic separationof these neurotransmitter derivatives was performed on Waters Acquity BEH (ethylene bridged hybrid) C18 column withparticle size of 1.7 μm at 35°C using a mobile phase consisting of 0.1 M acetate buffer (pH 6.0) and methanol (60:40, v/v) at a flowrate of 0.3 ml/min. The analytes were detected with the fluorescence detector (FLD) using derivatization with o-phthaldialdehyde(OPA), resulting in excitation at 340 nm and emission at 455 nm. Results: Several validation parameters including linearity(0.999), accuracy (101.1%), intra-day precision (1.52–1.84%), inter-day precision (2.47–3.12%), limit of detection (5–30 ng/ml)and quantification (100 ng/ml) were examined. The developed method was also used for the determination of these neurotransmittersin homogenates of selected rat brain structures. Conclusions: The presented UPLC-FLD is characterized by shorter separationtime (3.5 min), which is an adaptation of the similar HPLC methods and is an alternative for more expensive references techniquessuch as liquid chromatography coupled with tandem mass-spectrometry (LC-MS/MS) methods. Med Pr 2017;68(5):583–591