Optimization of ultra-performance liquid chromatography (UPLC) with fluorescence detector (FLD) method for the quantitative determination of selected neurotransmitters in rat brain
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Medical University of Lodz, Łódź, Poland (Faculty of Pharmacy, Department of Toxicology)
Nofer Institute of Occupational Medicine, Łódź, Poland (Department of Biological and Environmental Monitoring)
Online publication date: 2017-07-21
Corresponding author
Joanna Stragierowicz   

Medical University of Lodz, Faculty of Pharmacy, Department of Toxicology, Muszyńskiego 1, 90-151 Łódź, Poland
Med Pr 2017;68(5):583–591
Background: Glutamate (Glu) and γ-aminobutyric acid (GABA) are the main neurotransmitters in the central nervous system for excitatory and inhibitory processes, respectively. Monitoring these neurotransmitters is an essential tool in establishing pathological functions, among others in terms of occupational exposure to toxic substances. Material and Methods: We present modification 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 separation of these neurotransmitter derivatives was performed on Waters Acquity BEH (ethylene bridged hybrid) C18 column with particle 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 flow rate 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 neurotransmitters in homogenates of selected rat brain structures. Conclusions: The presented UPLC-FLD is characterized by shorter separation time (3.5 min), which is an adaptation of the similar HPLC methods and is an alternative for more expensive references techniques such as liquid chromatography coupled with tandem mass-spectrometry (LC-MS/MS) methods. Med Pr 2017;68(5):583–591