Capillary reversed-phase high-performance liquid chromatographic determination of acetyl-methylprednisolone in feline spinal cord.

Abstract

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) was used to determine acetylmethylprednisolone (A-MP) that had been 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 isocratic mobile phase of 40 mM triethylamine formate (TEAF, pH 3.2)-acetonitrile (50:50, v:v). The chromatographic behavior of A-MP was evaluated with respect to peak-area and peak-height by varying the A-MP concentration (12-190 microM) with a fixed injection volume (1 microliter), and by varying the injection volume (1-10 microliter) with a fixed concentration (12 microM) 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 directly spiked into feline spinal cord segments were solvent extracted, separated, and plotted against peak-area (r2 = 1.00). Background tissue 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 was confirmed via its molecular ion and corresponding product ions that were obtained by fast-atom bombardment tandem mass spectrometry (FAB-MS-MS).