title: Development and application of a UHPLC-MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum.
abstract: A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11-deoxycortisol, 11-deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra-high-performance liquid chromatography-tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra-high-performance liquid chromatography-tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922-0.9986, and the limits of detection and limits of quantification were in the range of 0.002-2 and 0.0055-5.5 ng ml-1, respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml-1) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml-1free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25-51.26 ng ml-1) and cortisol (range 32.57-52.24 ng ml-1), followed by 17-OH-pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.
authors: School of Pharmacy, University of Camerino, Via Sant' Agostino 1, 62032, Camerino, Italy.
authors: Michele
authors: 0000-0002-1287-0826
authors: M
authors: Genangeli
authors: School of Pharmacy, University of Camerino, Via Sant' Agostino 1, 62032, Camerino, Italy.
authors: Giovanni
authors: G
authors: Caprioli
authors: School of Pharmacy, University of Camerino, Via Sant' Agostino 1, 62032, Camerino, Italy.
authors: Manuela
authors: M
authors: Cortese
authors: School of Biosciences and Veterinary Medicine, University of Camerino, Via Circonvallazione 93/95, 62024, Matelica, Italy.
authors: Fulvio
authors: F
authors: Laus
authors: Eureka Lab Division, Via E. Fermi 25, 60033, Chiaravalle, Ancona, Italy.
authors: Mara
authors: M
authors: Matteucci
authors: School of Pharmacy, University of Camerino, Via Sant' Agostino 1, 62032, Camerino, Italy.
authors: Riccardo
authors: R
authors: Petrelli
authors: School of Pharmacy, University of Camerino, Via Sant' Agostino 1, 62032, Camerino, Italy.
authors: Massimo
authors: M
authors: Ricciutelli
authors: School of Pharmacy, University of Camerino, Via Sant' Agostino 1, 62032, Camerino, Italy.
authors: Gianni
authors: G
authors: Sagratini
authors: Eureka Lab Division, Via E. Fermi 25, 60033, Chiaravalle, Ancona, Italy.
authors: Stefano
authors: S
authors: Sartori
authors: School of Pharmacy, University of Camerino, Via Sant' Agostino 1, 62032, Camerino, Italy.
authors: Sauro
authors: S
authors: Vittori
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004300:Doping in Sports; D005260:Female; D006736:Horses / Q000097:blood*; D057230:Limit of Detection; D008297:Male; D055442:Metabolome; D013256:Steroids / Q000097:blood*; D015813:Substance Abuse Detection / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D013256:Steroids
keywords: UHPLC-MS/MS; equine serum; hormones; steroidal hormones; validation