Determination of 5-Hydroxytryptamine, Norepinephrine, Dopamine and Their Metabolites in Rat Brain Tissue by LC–ESI–MS–MS

Abstract

A liquid chromatography–electrospray ionization tandem mass spectrometry method has been developed to perform the determination of 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites, i.e., 5-hydroxyindole-3-acetic acid (5-HIAA), 4-hydroxy-3-methoxyphenylglycol (MHPG) sulfate, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat brain tissue. Analytes were separated on a Thermo C₁₈ column (4.6 mm × 250 mm, 5 μm, SN: 1245575T, Thermo electron corporation, USA) with a mobile phase of 0.05% formic acid/acetonitrile (92:8 for ESI⁺, 82:18 for ESI⁻, v/v) at the flow-rate of 0.8 mL min⁻¹. The LC system was coupled to a Waters Micromass Quattro Premier XE tandem quadruple mass spectrometer. MS acquisition of 5-HT, NE and DA was performed in positive electrospray ionization multiple reaction monitoring (MRM) mode, while negative electrospray ionization MRM mode was used to monitor their metabolites. The calibration curves were linear within the concentration range of 4–4,450 ng mL⁻¹ for 5-HT, 4–4,110 ng mL⁻¹ for NE and 4–4,100 ng mL⁻¹ for DA (r ≥ 0.999). The limit of quantitation was 4 ng mL⁻¹. 5-HIAA, MHPG, DOPAC and HVA have good linearity within the range of 12–1,000 ng mL⁻¹(r ≥ 0.998) and the limit of quantitation was 12 ng mL⁻¹. The intra- and inter-day RSD were lower than 8.45%. The method is sensitive, fast, accurate and usable for quantity determination of monoamine neurotransmitters and their metabolites in neuropsychiatric diseases.