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Volume 8, Issue 2 (Suppl) Chem Sci J 2017
ISSN: 2150-3494 CSJ, an open access journal Euro Chemistry 2017
May 11-13, 2017
May 11-13, 2017 Barcelona, Spain
4th European Chemistry Congress
L Molina-García et al., Chem Sci J 2017, 8:2(Suppl)
Optimization and validation of non-invasive HPLC-MS/MS method for stress quantification in free-living
ruminants
L Molina-García1, J M Pérez1, M Sarasa2, B Ureña-Gutiérrez1, J Espinosa1 and C Azorit1
University of Jaen, Spain
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 (FCM) determination
has proven to be a powerful tool. That is, high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-
MS/MS) has emerged as the most accurate method avoiding problems related to the non-specificity and matrix effects of the so-used
immunoassays. In this work we have optimized, developed and validated a reliable method for 11-ketoetiocholanolone (11-k), a
cortisol metabolite, quantification in ruminant’s fecal samples by using and HPLC-MS/MS method. An appropriate 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 subsequent clean-up with hexane, followed by purification and pre-concentration of targeted
metabolite with solid phase extraction (SPE). The final extract obtained was then analyzed 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 rigorous 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, referred to freeze-dried sample, were 13 and 40 μg kg-1, respectively. Recoveries in the range of 85-110% and RSDs not
higher than 15%for the complete analytical procedure, including extraction and analysis, were achieved.