Determination of monoamine neurotransmitters in zebrafish (Danio rerio) by gas chromatography coupled to mass spectrometry with a two-step derivatization

A sensitive analytical method for the determination of monoamine neurotransmitters (MNTs) in zebrafish larvae was developed using gas chromatography coupled to mass spectrometry. Six MNTs were selected as target compounds for neurotoxicity testing. MNTs underwent a two-step derivatization with hexamethyldisilazane (HDMS) for O-silylation followed by N-methyl-bis-heptafluorobutyramide (MBHFBA) for N-perfluoroacylation. Derivatization conditions were optimized by an experimental design approach. Method validation showed linear calibration curves (r 2 > 0.9976) in the range of 1–100 ng for all the compounds. The recovery rates were between 92 and 119%. The method was repeatable and reproducible with relative standard deviations (RSD) in the range of 2.5–9.3% for intra-day and 4.8–12% for inter-day variation. The limits of detection and the limits of quantitation were 0.4–0.8 and 1.2–2.7 ng/mL, respectively. The method was successfully applied to detect and quantify trace levels of MNTs in 5-day-old zebrafish larvae that were exposed to low concentrations of neurotoxic chemicals such as pesticides and methylmercury. Although visual malformations were not detected, the MNT levels varied significantly during early zebrafish development. These results show that exposure to neurotoxic chemicals can alter neurotransmitter levels and thereby may influence early brain development.