Simultaneous determination of prednisolone acetate, prednisolone, prednisone, cortisone and hydrocortisone in swine plasma using solid-phase and liquid-liquid extraction techniques

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of prednisolone acetate (PA), prednisolone (PO), prednisone (PN), cortisone and hydrocortisone in swine plasma is described. Extraction of the steroid mixture from swine plasma with dexamethasone as internal standard was accomplished by solid-phase extraction (SPE) or the more traditional liquid-liquid extraction (LLE) techniques. These compounds were analyzed by normal-phase HPLC with ultraviolet detection. Although a detectable sensitivity of 5 ng/ml is achieved by the SPE technique, the practical sensitivity is established as 10 ng/ml. Conversely, the practical sensitivity is 5 ng/ml for all compounds by the LLE technique. Calibration curves were found to be linear between 10 and 500 ng/ml by the SPE technique and between 5 and 100 ng/ml by the LLE technique. The average recovery of the steroids PA, PO and PN at 20 ng/ml is between 70 and 90%. PA is stable for up to 3 h in swine plasma at room temperature (22 degrees C) but is completely converted to PO within 24 h. PA is stable in swine plasma in an ice bath for over 24 h. The usefulness of this analytical technique is demonstrated by the intraperitoneal administration of 125 mg of PA to swine and the quantitative determination of PA, PO and PN in the plasma as a function of time.