Analysis of 17 neurotransmitters, metabolites and precursors in zebrafish through the life cycle using ultrahigh performance liquid chromatography-tandem mass spectrometry.
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 11ngmL(-1) and limits of quantification from 0.1 to 38ngmL(-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 (R(2)) 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.
Abbott Nutrition, R&D, Abbott Laboratories, Camino de Purchil 68, E-18004 Granada, Spain; Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry, University of Granada, Campus of Fuentenueva, E-18071 Granada, Spain. Electronic address: angela.santos@abbott.com.
A
A
Santos-Fandila
Abbott Nutrition, R&D, Abbott Laboratories, Camino de Purchil 68, E-18004 Granada, Spain.
E
E
Vázquez
Abbott Nutrition, R&D, Abbott Laboratories, Camino de Purchil 68, E-18004 Granada, Spain.
A
A
Barranco
Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry, University of Granada, Campus of Fuentenueva, E-18071 Granada, Spain.
A
A
Zafra-Gómez
Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry, University of Granada, Campus of Fuentenueva, E-18071 Granada, Spain.
A
A
Navalón
Abbott Nutrition, R&D, Abbott Laboratories, Camino de Purchil 68, E-18004 Granada, Spain.
R
R
Rueda
Abbott Nutrition, R&D, Abbott Laboratories, Camino de Purchil 68, E-18004 Granada, Spain.
M
M
Ramírez
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008018:Life Cycle Stages; D018377:Neurotransmitter Agents / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D015027:Zebrafish
D018377:Neurotransmitter Agents
Analytical method validation; Life cycle; Neurotransmitters; UHPLC–MS/MS; Zebrafish