Gopika Radhakrishnan, Vebjørn Larsen, Joar Breivik, Øystein Sæle, Pedro Araujo
Cleaner fish are widely used as a biological measure for controlling salmon lice in aquaculture. However, their production is frequently affected by developmental and physiological disorders, many of which may be associated with stress responses induced by husbandry-related factors. Analysis of stress-related hormones, such as cortisol and melatonin, in larval head and body tissues can therefore provide biochemical insight into the mechanisms underlying these production challenges. In this study, a simple two-solvent liquid-liquid extraction (LLE) and two standard addition approaches, without and with stable-isotope dilution, hereafter referred to as SAM and SAM+IS, respectively, were validated and compared for the quantitative LC-MS/MS determination of cortisol and melatonin in ballan wrasse (Labrus bergylta) larvae. Validated LLE-LC-MS/MS enabled matrix-compatible simultaneous quantification of both hormones in the selected tissues using the SAM and SAM+IS approaches. The calibration approaches were selective for the analytical hormones in head and body matrices. Considerable matrix effects were observed in most analytical systems, except for cortisol determination in head and body extracts using SAM+IS. All calibration models showed consistent linearity over the evaluated concentration range of 0-150 ng/mL, with correlation coefficients ranging from 0.993 to 0.999, indicating strong associations between spiked concentrations and analytical responses. Comparison of SAM and SAM+IS demonstrated that both approaches provided comparable estimates of endogenous cortisol and melatonin concentrations in head and body extracts. Application of the SAM+IS method to larvae reared under two different commercial feeding strategies revealed that developmental stage had a significant tissue-specific effect on cortisol and melatonin levels, independent of feeding strategy.