PMID- 30377495
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20230928
IS  - 2045-7758 (Print)
IS  - 2045-7758 (Electronic)
IS  - 2045-7758 (Linking)
VI  - 8
IP  - 18
DP  - 2018 Sep
TI  - HPLC-QTOF method for quantifying 11-ketoetiocholanolone, a cortisol metabolite, 
      in ruminants' feces: Optimization and validation.
PG  - 9218-9228
LID - 10.1002/ece3.4285 [doi]
AB  - Studies of animal ecology can benefit from a quantified understanding of 
      eco-physiological processes and, in particular, of the physiological responses in 
      free-ranging animals to potential stressors. The determination of fecal cortisol 
      metabolites as a noninvasive method for monitoring stress has proved to be a 
      powerful tool. High-performance liquid chromatography coupled with tandem mass 
      spectrometry (HPLC-MS/MS) has emerged as the most accurate method for avoiding 
      problems related to the nonspecificity of immunoassays. In this study, we 
      optimize and validate a reliable method using HPLC-MS/MS for quantifying 
      11-ketoetiocholanolone (11-k), a representative fecal cortisol metabolite in 
      ruminants. An appropriate extraction and purification procedure was developed 
      taking into account the complex nature of feces. The final extract obtained was 
      then analyzed with HPLC-MS/MS using a quadrupole-time-of-fly (QTOF) tandem mass 
      spectrometer with an electrospray ionization interface operating in positive 
      mode, which allowed an unequivocal determination of the metabolite due to its 
      accurate mass capabilities. After rigorous optimization of both sample extraction 
      and the HPLC-QTOF parameters, making use of feces from free-ranging Iberian ibex, 
      ideal conditions were established. Matrix-matched standards were used to 
      calibrate the method. The limit of detection and quantification was 13- and 40- 
      ng/g, respectively. The validation of the method was performed with recoveries in 
      the range of 85-110%, a figure much higher than the 60% obtained with the 
      previous extraction methods used in our laboratory, and with relative standard 
      deviations (RSDs) no higher than 15% for the complete analytical procedure, 
      including extraction and analysis. The time required for the fecal 11-k analysis 
      was greatly reduced in comparison with the previous work carried out in our 
      laboratory. This is the first time that QTOF mass detection coupled with HPLC has 
      been validated for 11-k quantification in feces from free-ranging ruminants such 
      as Iberian ibex. Given the high selectivity and sensitivity attained, our method 
      could become a useful tool for noninvasive stress quantification in ruminants.
FAU - Molina-Garcia, Lucia
AU  - Molina-Garcia L
AUID- ORCID: 0000-0001-6830-9184
AD  - Department of Animal and Plant Biology, and Ecology University of Jaen Jaen 
      Spain.
FAU - Perez, Jesus M
AU  - Perez JM
AUID- ORCID: 0000-0001-9159-0365
AD  - Department of Animal and Plant Biology, and Ecology University of Jaen Jaen 
      Spain.
FAU - Sarasa, Mathieu
AU  - Sarasa M
AUID- ORCID: 0000-0001-9067-7522
AD  - Federation Nationale des Chasseurs (FNC) Issy-les-Moulineaux France.
AD  - Present address: BEOPS Toulouse France.
FAU - Urena-Gutierrez, Benjamin
AU  - Urena-Gutierrez B
AD  - Department of Animal and Plant Biology, and Ecology University of Jaen Jaen 
      Spain.
FAU - Espinosa, Jose
AU  - Espinosa J
AD  - Department of Animal and Plant Biology, and Ecology University of Jaen Jaen 
      Spain.
FAU - Azorit, Concepcion
AU  - Azorit C
AUID- ORCID: 0000-0001-9538-7110
AD  - Department of Animal and Plant Biology, and Ecology University of Jaen Jaen 
      Spain.
LA  - eng
PT  - Journal Article
DEP - 20180801
PL  - England
TA  - Ecol Evol
JT  - Ecology and evolution
JID - 101566408
PMC - PMC6194299
OTO - NOTNLM
OT  - 11-ketoetiocholanolone
OT  - Capra pyrenaica
OT  - HPLC-QTOF
OT  - fecal cortisol metabolites
OT  - noninvasive monitoring
OT  - ruminant stress
OT  - solid phase extraction
EDAT- 2018/11/01 06:00
MHDA- 2018/11/01 06:01
PMCR- 2018/09/01
CRDT- 2018/11/01 06:00
PHST- 2017/06/08 00:00 [received]
PHST- 2018/04/23 00:00 [revised]
PHST- 2018/05/19 00:00 [accepted]
PHST- 2018/11/01 06:00 [entrez]
PHST- 2018/11/01 06:00 [pubmed]
PHST- 2018/11/01 06:01 [medline]
PHST- 2018/09/01 00:00 [pmc-release]
AID - ECE34285 [pii]
AID - 10.1002/ece3.4285 [doi]
PST - epublish
SO  - Ecol Evol. 2018 Aug 1;8(18):9218-9228. doi: 10.1002/ece3.4285. eCollection 2018 
      Sep.