Identification of cortisol metabolites with LC-MS/MS in plasma, skin mucus, bile and faeces for stress evaluation of farmed Atlantic salmon.

As a stress hormone, cortisol and more recently its metabolites are analysed when assessing fish stress and welfare status, although the exact identity of these metabolites is not clearly defined for the Atlantic salmon. LC-MS/MS techniques, owing to their specificity, sensitivity and ability to simultaneously identify and measure several relevant compounds, can be useful tools for this purpose. Using the guidelines provided by the European Decision no. 657/2002/EC for validation, the LC-MS/MS method presented here, can reliably identify and quantify cortisol and five of its metabolites (5ß-THF, cortisone, 5ß-DHE, 5ß-THE and ß-cortolone) in bile and faeces, and cortisol and cortisone in skin mucus and blood plasma of farmed Atlantic salmon within 15 min. Identified as the most predominant compound in faeces and bile, 5ß-THE is proposed as a candidate stress biomarker when using these matrices. A decision limit (CCα) below 5 ng/mL, a detection capability (CCß) and a limit of detection (LOD) below 10 ng/mL and a limit of quantitation (LOQ) below 30 ng/mL were typically obtained for most of the compounds. The concentrations of these compounds measured in either non-stressed or stressed fish were all above the CCα, CCß, LOD and the LOQ of the method. The latter consequently demonstrated significant difference in cortisol metabolites concentrations between the two groups of fish. The present study further demonstrates that pooling of samples from several individuals could provide reliable results for farmed fish stress evaluation, when sample materials are insufficient in terms of quantity.