Development of a LC-MS-MS method for the simultaneous determination of natural steroidal hormones and synthetic anabolic steroids in equine and bovine blood matrix

Monitoring the hormones in equine matrix is crucial to understanding the health status of horses , and also to monitoring the abuse of these substances before a race or a horse transaction. Few analytical procedures exist which are “self made methods”, generally based on immunoassay analysis, often impossible to replicate in other laboratories or too complex for the staff. On that, it is necessary to develop new sensitive analytical methods, that can be diffused to external vets and give correct and comparable results. Furthermore, the few analytical procedures involving an HPLC-MS-MS system reported in literature allow for simultaneous quantification of no more than three or four hormones [1][2]. The proposed method makes it possible to detect and quantify seventeen hormones and metabolites in a single assay, in just ten minutes. Quantifiable hormones with the proposed method are: Pregnenolon, 17-OH-Pregnenolon, Progesteron, 17-OH-Progesteron, Androsteron, Androstenedion, DHEA, DHEAS, Testosteron, Cortisol, Corticosteron, Aldosteron, 11-Deossicortisol, 11-Deossicorticosteron, Diidrotestosteron, Estron, Estradiol. A deuterated hormone ( Testosteron-D3) has been used as internal standards in order to set a more accurate and precise procedure. The method was developed using the Agilent UHPLC chromatographic system (1290) using a Zorbax RRHD C18 – 1,8 μm column and a 6420 Agilent Mass Spectrometer . The procedure is fast and intuitive; the sample preparation is very easy, with the serum deproteinized in vials using a deproteinizing solution, centifuged and injected into the system. The mobile phases are made of water and acetonitrile, both containing formic acid. Overall, the method is very simple and robust and it brings about a remarkable saving of time and money with respect to previously reported methods; in fact, using the UHPLC–Tandem Mass spectrometer enables simultaneous quantification of seventeen Steroidal Hormones. The method has been applied to a number of equine and bovine blood samples supplied from different breeding farms and territorial vests