Liquid chromatographic analysis of prednisolone, prednisone and their 20-reduced metabolites in perfusion media

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20α-dihydro and 20β-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6α-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0–1 μg/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20α-dihydroprednisone) to 8.8% (20β-dihydroprednisolone) and 4.1% (20β-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20α-dihydroprednisone) to 9.1% (20β-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C₁₈ columns which were linked together and maintained at 40°C.