Optimization and validation of non-invasive HPLC-MS/MS method for free-living ruminants stress quantification

4^th European Chemistry Congress

May 11-13, 2017 Barcelona, Spain

L Molina-Garcia, J M Perez, M Sarasa, B Urena-Gutierrez, J Espinosa1 and C Azorit

University of Ja�©n, Spain
Federation Nationale des Chasseurs (FNC), France

Posters & Accepted Abstracts: Chem Sci J

Abstract :

Wildlife management and conservation can benefit from a quantified understanding of physiological response of free-ranging animals to the various potential stressors. Non-invasive stress monitoring by fecal cortisol metabolites determination has probed to be a powerful tool. That is, high performace liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/ MS) has emerged as the most accurate method avoiding problems related to the inespecificity and matrix effects of the so-used immunuassays. In this work we have optimized, developed and validated a reliable method for 11-ketoetiocholanolone (11-k), a cortisol metabolite, quantification in ruminantâ��s fecal samples by using and HPLC-MS/MS method. An appropiate extraction and purification procedure was developed to take into account the complex nature of feces. The method consisted in a primary fecal samples extraction with methanol and subsequently clean-up with hexane, followed by purification and preconcentration of targeted metabolite with solid phase extraction (SPE). The final extract obtained was then anlyzed by HPLC-MS/MS making used of a quadrupole-time-of-fly (Q-TOF) tandem mass spectrometer with an electrospray ionization interface operating in positive mode. An isotope internal standard was used in order to minimize matrix effect and to compensate the alterations of the analytical signal. After a rigurous optimization of both sample extraction and HPLC-QTOF parameters, the method was satisfactory validated and the best conditions were stablished. Matrix-matches standards were used for the calibration of the method. The limit of detection and quantification, refered to freeze-dried sample, were 13 and 40 �¼g kg-1, respectively. Recoveries in the range of 85-110% and RSDs not higher than 15% for the complete analytical procedure, incluiding extraction and analysis, were achieved. For the best of our knowledge, this is the first time that the hybrid Q-TOF mass detection coupled to HPLC has been employed for the fecal 11-k quantification in ruminants. Due to the high specificity and sensitivity achieved, the method developed could be used as standard technique for unequivocal fecal 11-k quantification not only in ruminants but also in other related species. In addition, the evaluation of inter-individual differences of stress response could be carried out in the future in an accurate manner by using the present method, thus reducing the effects of potential confounding factors. lmgarcia@ujaen.es