Rapid analysis of neurotransmitters in rat brain using ultra‐fast liquid chromatography and tandem mass spectrometry: application to a comparative study in normal and insomnic rats

Abstract

Neurotransmitters and their metabolites in central nervous system were known to play a significant role in sedation and hypnosis. A rapid and sensitive UFLC-MS/MS method for simultaneous determination of serotonin, 5-hydroxyindole acetic acid (5-HIAA), tryptophan (Try), dopamine (DA), norepinephrine (NE), γ-aminobutyric acid (GABA), glutamic acid (Glu) and acetylcholine (Ach) in rat brain without derivatization, ion-pairing reagent or pre-concentration was developed. Analytes and IS were separated on a Inertsil ODS-EP column (150 mm × 4.6 mm, 5 µm particles) and analyzed in a single chromatographic run in less than 9.0 min, using gradient elution with the mobile phase consisting of methanol and 0.01% acetic acid in water at a flow rate of 1.2 ml min(-1) . The detection of the analytes was performed on 4000Q UFLC-MS/MS system with turbo ion spray source in positive ion and multiple reaction monitoring mode. The developed method provided excellent linear calibration curves for the assay of analytes (R(2) ≥ 0.9915). Limits of quantification were in the range of 1.0 ng ml(-1) to 1.0 µg ml(-1) for the analytes in rat brain. Intra- and inter-day precision and accuracy of analytes were well within acceptance criteria (15%). Mean extraction recoveries of analytes and IS from rat brain were all more than 80.0%. Furthermore, the validated method was successfully applied to comparing profiles of analytes in normal and insomnic rat brains. Results indicated that there were statistically significant differences for serotonin, 5-HIAA, DA, NE, Glu and Ach, but no significant difference for Try and GABA between two groups.