Cortisol (hydrocortisone) is a steroid hormone produced by the adrenal gland. Hydrocortisone is a hydrophobic neutral compound with some polar groups in its structure. Several Primesep mixed-mode columns were used for separation of hydrocortisone and its degradation products. Each column showed a different selectivity for separation, which attributed to different polar-embedded groups on Primesep 100 and Primesep D columns. Both columns can be used for degradation studies of various hydrophobic compounds.