Analysis of 17 neurotransmitters, metabolites and precursors in zebrafish through the life cycle using ultrahigh performance liquid chromatography–tandem mass spectrometry

• Analysis of 17 neurotransmitters, metabolites and precursors using UHPLC–MS/MS.
• The sample treatment is a simple deproteinization using methanol and formic acid.
• Validation of the method: quality parameters, accuracy (trueness and precision).
• Analysis of compounds in zebrafish from early stages of development to adulthood.
• The time-course of the neurocompounds through the life cycle of the animal is showed.

An ultrahigh performance liquid chromatography–tandem mass spectrometry method for the identification and quantification of neurotransmitters, metabolites and precursors at different stages in zebrafish life was developed. Betaine, glutamine, glutamic acid, γ-aminobutyric acid, phosphocholine, glycerophosphocholine, cytidine 5′-diphosphocholine, choline, acetylcholine, dopamine, norepinephrine, serotonin, tyrosine, epinephrine, tryptophan, 5-hydroxyindolacetic acid and agmatine were selected as analytes. The method consisted of a simple deproteinization of samples using methanol and formic acid, subsequent injection onto the chromatographic equipment and quantification with a triple quadrupole mass spectrometer detector using an electrospray ionization interface in positive mode. Limits of detection ranged from 0.02 to 11 ng mL−1 and limits of quantification from 0.1 to 38 ng mL−1, depending on the analyte. The method was validated according to US Food and Drugs Administration (FDA) guideline for bioanalytical assays. Precision, expressed as relative standard deviation (%RSD), was lower than 15% in all cases, and the determination coefficient (R2) was equal or higher than 99.0% with a residual deviation for each calibration point lower than ±25%. Mean recoveries were between 85% and 115%. The method was applied to determine of these compounds in zebrafish from early stages of development to adulthood and showed the time-course of neurotransmitters and others neurocompounds through the life cycle. The possibility of measuring up to 17 compounds related with the main neurotransmitter systems in a simple analytical method will complement and reinforce the use of zebrafish in multiple applications in the field of neurosciences. The proposed method will facilitate future studies related with brain development.