High-performance liquid chromatographic assay for methylprednisolone and its soluble prodrug esters in dog plasma

A high-performance liquid chromatographic method with ultraviolet detection (lambda max = 243 nm) has been developed for the simultaneous determination of methylprednisolone (MP) and its water-soluble prodrug esters methylprednisolone hemisuccinate (MPS) and N,N,N'-triethylethylenediamine amide of 6 alpha-methylprednisolone-21-hemisuberate hydrochloride (TMPS) in dog plasma. A reversed-phase liquid chromatographic separation was performed on a Microsorb C8 (3 microns) column equipped with a C8 5-microns guard column. The mobile phase composition was water--acetonitrile--methanol--dimethyloctylamine--acetic acid (65.5:34:0.4:0.04:0.04). The methyl ester of phenethylcarbamate was employed as an internal standard. The chromatographic responses were linear up to 25 micrograms/ml for MP, 70 micrograms/ml for MPS, and 95 micrograms/ml for TMPS. The sensitivity of the assay by ultraviolet detection is approximately 4, 8, and 12 ng/ml of plasma for MP, MPS and TMPS, respectively. The assay variability in terms of 95% confidence limit for each steroid is less than 4.5%. Plasma concentration--time curves are reported for MP, MPS, and TMPS after intravenous administration of MPS and TMPS equivalent to 3, 10 and 30 mg MP per kg body weight of dog. The assay methodology is simple, selective and reproducible for the quantitative determination of MP, MPS and TMPS in dog plasma.