Ultra-sensitive UHPLC-MS/MS method for simultaneous quantification of 31 neurochemicals in zebrafish larvae and brain.

The precise determination of neurotransmitters and their metabolites in biological matrices is critical for research on neurological disorders, including those originated by the exposure to neurotoxic chemicals. This study presents an optimized liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for 31 neurochemicals, including neurotransmitters, their metabolites and precursors. The method is aimed at achieving lower limits of detection (LOD) and quantification (LOQ) compared to those currently available, while simultaneously expanding the number of compounds analyzed. The proposed approach achieves instrumental detection limits (IDLs) from 0.02 to 7.2 pg, except for homovanillic acid showing a value of 150 pg, with recovery values across all target analytes comprised between 50 and 140 % in zebrafish larvae and 55 and 150 % in adult zebrafish brain, when using internal standard calibration. Overall, the optimized method represents a significant improvement over previous methods, making it robust, accurate and sensitive enough for applications in studies requiring detailed analysis of neurochemicals in complex biological samples, such as zebrafish larvae and adult zebrafish brain. Adult zebrafish brain samples from adults intraperitoneally exposed to 5 doses of 1-methyl-4-phenyl,6-tetrahydropyridine (MPTP, 100 mg/kg body weight (bw)) were accurately analyzed using the optimized UHPLC-MS/MS method, which provided insights into neurochemical alterations induced by this neurotoxin.