An LC-MS/MS-based approach for monitoring monoaminergic status in lizard brains: method development and real-samples...

Monoamines play essential roles in regulating brain functions, influencing behaviours and physiological processes. Despite significant advances in mammalian models, studies on reptiles are limited, restricting our understanding of their neurochemical and behavioural interactions. This study aimed to develop and validate a selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 11 monoamines and their metabolites (adrenaline, dopamine, levodopa, homovanillic acid, 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, serotonin, norepinephrine, tryptophan, tyrosine, vanillylmandelic acid) in lizard brains, examining interspecies and sex-specific variations and their potential link to behavioural differences between Podarcis siculus and Podarcis melisellensis. Brain samples from 132 lizards were analysed using the optimized LC-MS/MS method, which included rigorous pre-analytical preparation to ensure analyte stability. The method demonstrated high sensitivity and specificity for monoamines, with the limit of quantitation lower than 5 ng mL^-1. Statistical analyses were performed to assess species, sex, and interaction effects. Significant species differences were observed in tryptophan, 5-hydroxytryptophan and 5-hydroxyindoleacetic acid, with P. siculus males exhibiting the highest levels. No differences were found in the dopamine, noradrenaline or adrenaline pathways. Serotonergic differences align with previously recorded behavioural differences between these two species. This is the first comprehensive study profiling monoamines in lizard brains using LC-MS/MS, providing insights into species-specific neurochemical patterns. The findings underscore the utility of lizards as comparative models in neuroscience and highlight the need for further research into the interplay between neurochemistry and behaviour.