Development and application of an UHPLC-MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum

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, DHEA, DHEAS, Testosterone, Cortisol, Corticosterone, Aldosterone, 11-Deoxycortisol, 11-Deoxycorticosterone, Dihydrotestosterone, Estrone, Estradiol) has been developed in equine serum using the UHPLC-MS/MS technique. 400 μL of sample were deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in UHPLC-MS/MS 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 (CV) 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 (LODs) and limits of quantification (LOQs) were in the range of 0.002–2 ng ml-1 and 0.0055-5.5 ng ml-1, respectively. The detected LOQ for testosterone (i.e. 50 pg ml-1) is two-fold lower with respect to its threshold admitted in geldings plasma (100 pg ml-1 free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of 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.