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Abstract
A quantitative LC–ESI single quadrupole MS method for the determination of cortisol (F), cortisone (E), prednisolone (PL), and prednisone (PN) in bovine urine has been developed and validated. After adding flumethasone as internal standard, the samples were subjected to filtration, deconjugation, and solid-phase extraction, while the chromatographic separation was achieved using a Restek Ultra II Allure Biphenyl column with isocratic mobile phase. The analytes were detected after negative electrospray ionization using SIM mode. In order to obtain spectra with maximum intensities of at least one of the three characteristic ions, (M + formate)−, (M − H)−, and [(M − H) – CH2O]−, an individual optimization of MS parameters for each corticosteroid was set up. MS data was acquired in the three-ion selected monitoring mode and the ion ratios between chosen diagnostic ions were used in order to increase the specificity. Calibration graphs were linear and the intra-day and intermediate precision was estimated as RSD values which were less than 17%. For F and E, obtained values indicated negligible absolute matrix effects (103% and 98%, respectively). The method was applied to real samples, and basal levels of F and E were preliminarily evaluated, while PL and PN were not detected.
ACKNOWLEDGMENT
This work was supported by research grants from the FEASR - Programma di Sviluppo Rurale 2007-2013, Misura 124 entitled “Valorizzazione del prodotto carneo da bovino adulto a fine carrier” (responsible L. Chiesa) and from the “Bruno Maria Zaini” Reference Centre of Comparative Pathology, University of Torino, and from the Regione Piemonte, project 2009: “Prevenzione dell'uso di anabolizzanti in zootecnia: le bioteconologie nello sviluppo di disciplinari per la qualità e salubrità della carne e derivati” (responsible B. Biolatti).