Liquid chromatography–mass spectrometry method development for monitoring stress-related corticosteroids levels in pig saliva

• SPE + LC–MS/MS (ESI) method for corticoesteroids in pig saliva developed and validated.
• Factors studied at farm were: pig sex, breeds (Duroc, Pietrain & L62), and time (0–6 h).
• Maxima non-stress levels in different pig breeds at farm: 3.0 μg/L of cortisol in saliva.
• Quantification limits from 0.050 to 0.30 μg/L for the enrichment of 1.0 mL saliva.
• Recoveries between 60 and 90% (with RSDs < 11%) for five corticoesteroids.

Biochemical response stressors results in an increase of adrenocortical activity. Before knowing the corticosteroid levels in saliva in a stressful situation, baselines salivary levels should be established. A method for simultaneous determination of five corticosteroids was developed, validated and applied to pig saliva at farms. The method employs solid-phase extraction (SPE) coupled with clean-up extraction step using silica cartridge in the same step followed by liquid chromatography/tandem mass spectrometry (LC–MS/MS), using electrospray ionization (ESI) in positive mode. The overall method quantification limits range from 0.050 to 0.30 μg/L for the enrichment of 1.0 mL saliva samples and analyte recoveries are between 60 and 90% (RSD < 11%). Some factors studied were: pig sex, breeds, and time at farm. The analytical method clearly shows that CRL and CRS levels of, respectively, 3.0 and 4.0 μg/L in saliva can be indicative of maxima non-stress levels in different pig breeds at farm.