Study of natural and artificial corticosteroid phase II metabolites in bovine urine using HPLC-MS/MS

Corticosteroid compounds are widely used therapeutically for their anti-inflammatory properties and sometimes as growth promoters in food producing animals. In the field of drug residue analysis, knowledge of the main metabolic pathways of target analytes improves the efficiency of the corresponding control. Thus, phase II metabolism of corticosteroids, for which very little literature is available, was investigated in cattle. An LC-MS/MS detection method was developed for five commercially available conjugated corticosteroids, permitting direct monitoring during the development of their separation on anion exchange SPE. This separation method is further applicable to other potential urinary conjugated corticosteroids. Because our purpose was not to identify all the existing corticosteroid phase II metabolites, but to obtain their total relative proportions, enzymatic hydrolysis was optimized and performed on each separated fraction (glucuronides and sulfates). Finally, the phase II metabolic profiles of natural and artificial corticosteroids in bovine urine were studied and compared. LC-MS/MS detection with negative electrospray ionization appeared efficient for both glucuronide and sulfate conjugated corticosteroids, and quaternary ammonium stationary phase permitted their effective separation. The experimental design used for optimization of the enzymatic hydrolysis with a purified Helix pomatia preparation demonstrated optimal values for pH 5.2, temperature of 50 degrees C and incubation duration of 4h. Results on bovine urine samples collected on two animals before and after dexamethasone administration showed important differences regarding the proportion of total conjugated forms between endogenous cortisol, endogenous tetrahydrocortisol, and exogenous dexamethasone. This proportion appeared significantly higher for tetrahydrocortisol (40-65%) than cortisol (2-8%) or dexamethasone (4-27%). This innovative methodology demonstrates the suitability of anion exchange SPE and LC-MS/MS for the study of steroid hormones phase II metabolism, and appears promising to investigate metabolic profile differences linked to the hormone administration mode or origin, with direct application in the field of doping controls.