title: Simultaneous determination of methylprednisolone and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt in human urine by high-performance liquid chromatography with ultraviolet detection.
abstract: A reversed-phase high-performance liquid chromatographic assay with ultraviolet detection at 243 nm has been developed for the quantitative determination of methylprednisolone (MP) and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt (MPSO) in human urine following therapeutic doses in humans. The assay procedure involves stabilization of urine samples by addition of disodium ethylenediaminetetraacetic acid (Na2EDTA) and ion-pair extractions of MPSO using tetraethylammonium chloride (TEACl) as the counter ion. After extracting both drugs and internal standard into chloroform, the extract was evaporated to dryness under nitrogen. The resulting residue was reconstituted in 200-500 microliters of mobile phase and chromatographed on an IBM C18 reversed-phase column (5 microns). The mobile phase was a mixture of water-acetonitrile-isopropanol (71.2:18.8:10.0, v/v) containing 75 microliters of 0.1 M hydrochloric acid and 0.450 g of TEACl per liter. Propyl p-hydroxybenzoate was used as an internal standard. The extraction efficiencies of MP and MPSO were greater than 90% using the ion-pairing agent TEACl. The chromatographic responses were linear up to about 200 micrograms/ml for MP and 80 micrograms/ml for MPSO and had sufficient precision and accuracy to provide quantitative data from human urine. The assay detection limit was about 8 ng/ml for MP and 25 ng/ml for MPSO in human urine. Stability studies in urine indicated that without Na2EDTA stabilization and at room temperature, rapid degradation of MPSO occurred in urine. Addition of EDTA to the urine specimen and storage at -70 degrees C increased the stability of MPSO, and little or no degradation was observed in urine stored for more than 60 days. The method has been used in the simultaneous determination of MP and MPSO in urine specimens obtained from a single-dose tolerance study of MPSO in normal male volunteers.
authors.affiliation: Drug Metabolism Research, Upjohn Company, Kalamazoo, MI 49001.
authors.forename: J A
authors.initials: JA
authors.lastname: Shah
authors.forename: D J
authors.initials: DJ
authors.lastname: Weber
mesh_terms: D000293:Adolescent; D000328:Adult; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004492:Edetic Acid; D006801:Humans; D008297:Male; D008775:Methylprednisolone / Q000031:analogs & derivatives* / Q000627:therapeutic use / Q000652:urine*; D008875:Middle Aged; D013056:Spectrophotometry, Ultraviolet; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds
chemical_list: D013757:Tetraethylammonium Compounds; D019789:Tetraethylammonium; D004492:Edetic Acid; C052305:methylprednisolone suleptanate; D008775:Methylprednisolone