CAPILLARY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF ACETYL-METHYLPREDNISOLONE IN FELINE SPINAL-CORD

Capillary reversed-phase high-performance liquid chromatography (RP-HP LC) was used to determine acetylmethylprednisolone (A-MP) that had bee n administered to feline spinal cord tissue. The method used a 300 mm x 0.32 mm I.D. packed capillary octadecylsilyl (ODS) column and an iso cratic mobile phase of 40 mM triethylamine formate (TEAF, pH 3.2)-acet onitrile (50:50, v:v). The chromatographic behavior of A-MP was evalua ted with respect to peak-area and peak-height by varying the A-MP conc entration (12-190 mu M) With a fixed injection volume (1 mu l), and by varying the injection volume (1-10 mu l) with a fixed concentration ( 12 mu M) of A-MP. The limit of detection (signal-to-noise ratio, 3:1) was 250 pg (600 fmol) of synthetic A-MP. Various amounts of A-MP direc tly spiked into feline spinal cord segments were solvent extracted, se parated, and plotted against peak-area (r(2) = 1.00). Background tissu e without A-MP gives minimal (< 1%) interference at 243 nm. The method also detects exogenous A-MP that was administered into feline spinal cord via an intrathecal injection, Furthermore, the presence of A-MP w as confirmed via its molecular ion and corresponding product ions that were obtained by fast-atom bombardment tandem mass spectrometry (FAB- MS-MS).