Abstract
Two reversed-phase high performance liquid chromatographic assays were developed for measuring methylprednisolone-hemisuccinate (MPHS) and metabolites in serum, urine and bile. The first used an acetonitrile:acetate buffer mobile phase to separate MPHS and its active metabolite methylprednisolone (MP), while the second resolved MPHS and subsequent metabolites of MP by a mobile phase containing methanol: citrate buffer: tetrahydrofuran. Internal standards used in quantification were 11-deoxy-17-hydroxy-corticosterone for the first and testosterone for the second methods. The steroids were extracted from body fluids using Extrelut columns and were detected by monitoring their U. V. absorption at a wavelength of 251 nm. Alternative detection principles involving either electrochemistry or derivatization with dansylhydrazine and fluorimetry were unsuccessful in detecting smaller quantities of MP. The percentage of a 1g dose of methylprednisolone sodium succinate (MPSS) that was excreted in urine by rheumatoid arthritis patients during the first 24 hours as unconjugated MPHS and MP was approximately 10 and 12%, respectively. Over the same period patients with glomerulonephritis excreted approximately 5% as MPHS and MP together. 20-alpha- (20a-) and 20-beta-hydroxymethylprednisolone (20b-OH-MP) were synthesized, and identified in urine collected following an infusion of MPSS, by gas chromatography-mass spectrometry (GC-MS). They accounted for a further 7 and 1% respectively, of a 1g dose of MPSS excreted during the first 24 hours. On average, 1% of the dose appeared in urine during the second 24 hour period as MPHS, MP, 20a- and 20b-OH-MP. Very few MPHS-related compounds (1%) were excreted as conjugates with glucuronic acid. Total urinary excretion of the four steroids was similar between rheumatoid arthritis and liver transplant patients. However, hydrolysis of MPHS to MP and subsequent metabolism of MP to 20a-OH-MP was impaired after liver transplantation. Four carboxylic acid metabolites of MP were identified in urine following MPSS, two of which were synthesized.