Improvement of conditions for the determination of neurotransmitters in rat brain tissue by HPLC with fluorimetric detection

Abstract

Gradient elution for the determination of six selected neurotransmitters (3,4-dihydroxyphenylacetic acid, dopamine, homovanillic acid, 5-hydroxyindole-3-acetic acid, L-noradrenaline, serotonin) by HPLC with fluorescence detection on the C18 reverse phase was designed and optimized. The elution used the binary mobile phase of aqueous solution of 12 × 10⁻³ mol dm⁻³ acetic acid, 0.26 × 10⁻³ mol dm⁻³ Na₂EDTA, pH = 3.5) and methanol. Furthermore, modified homogenization medium, prepared by mixing methanol and an aqueous solution (0.4 mol dm⁻³ perchloric acid, 6 × 10⁻³ mol dm⁻³ cysteine, 1 × 10⁻³ mol dm⁻³ L-ascorbic acid, and 0.54 × 10⁻³ mol dm⁻³ Na₂EDTA) in a ratio of 1:1 (v/v), was introduced in the preparation of rat brain tissue to increase the stability of neurotransmitters in the sample before the analysis. Both developed procedures were successfully applied to the determination of selected neurotransmitters in authentic brain tissue samples from rats affected by morphine and/or morphine abstinence. The statistically significant increases in dopamine, 3,4-dihydroxyphenylacetic acid, and serotonin levels in the striatum, as well as L-noradrenaline in the cerebral cortex of rats affected by intraperitoneal administration of morphine, were measured.

Graphical abstract