Prednisolone (P) is a corticosteroid derived from hydrocortisone with approximately 3 to 5 times the glucocorticoid and anti-inflammatory activity of hydrocortisone. Due to the potential for endogenous conversion of hydrocortisone to P, an international threshold of 10 micrograms per litre (mg/L) for free (i.e. non-conjugated) P in equine urine is used by racing authorities to distinguish between administered and naturally produced P.
The focus on free P has limited the diagnostic potential for monitoring phase II metabolites such as prednisolone glucuronide (PG) and prednisolone sulfate (PS). This study aimed to detect urinary PG and PS following oral administration of P in horses to provide corroborative evidence of in vivo metabolism of P.
Oral administration of P (1 g per day for 5 consecutive days) was performed with three Standardbred geldings. Urine samples were collected once prior to the first dose, then once daily on the final 3 days of treatment. Following the final oral dose, urine samples were collected at regular intervals up to 96 h.
Urinary free P was quantified from 1 mL sample using solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), which was validated to support the 10 mg/L threshold. Qualitative determination of PG and PS was performed using a modified SPE method and liquid chromatography-high resolution mass spectrometry (LC-HRMS).
The presence of PG and PS was confirmed following oral P administration. PG exhibited higher relative abundance compared to PS. Identification of both metabolites was supported by corresponding MS² fragmentation data.
This study demonstrated that qualitative monitoring of PG and PS can support the quantitative threshold of free P in equine urine.