High-performance liquid chromatographic determination of hydrocortisone and methylprednisolone and their hemisuccinate esters in human serum.

A high-performance liquid chromatographic method is described for the simultaneous determination of methylprednisolone (MP) and methylprednisolone hemisuccinate (MPHS), or hydrocortisone (HC) and hydrocortisone hemisuccinate (HCHS) in human serum. Reversed-phase liquid chromatography was performed on a microparticulate C18 column (Spherisorb, 5 micron) using a mobile phase of 2% glacial acetic acid, 30--35% acetonitrile, 70--65% water with ultraviolet detection (254 nm). The method uses 17 alpha-hydroxyprogesterone as the internal standard for the determination of methylprednisolone and its hemisuccinate ester, or 11-deoxy-17-hydroxycorticosterone as the internal standard for the determination of hydrocortisone and its hemisuccinate ester. The sensitivity is 0.03 microgram/ml for HC, 0.07 microgram/ml for MP, 0.04 microgram/ml for MPHS, and 0.10 microgram/ml for HCHS, with a detection limit of 0.02 microgram/ml for all four steroids. Calibration curves are linear up to 3 micrograms/ml for MP or MPHS (as equivalent MP) and up to 4 micrograms/ml for HC and 7 micrograms/ml (as equivalent HC) for HCHS. The pooled relative standard deviation for replicate for each steroid is less than 7%. Plasma concentration--time curves are reported for MP and MPHS or HC and HCHS of two human subjects following intramuscular administration of 125 mg of methylprednisolone sodium succinate for injection, U.S.P., or 250 mg of hydrocortisone sodium succinate for injection, U.S.P.