HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR DETECTION OF PHYSOSTIGMINE AND ESEROLINE IN PLASMA USING A SILICA-GEL COLUMN AND A PERCHLORIC-ACID MOBILE PHASE

A high-performance liquid chromatographic (HPLC) procedure that employs a silica gel column, a perchloric acid in methanol mobile phase and fluorescence detection has been developed for the determination of the concentrations of physostigmine and its metabolite, eseroline, in plasma. The method requires plasma samples to contain an ascorbic acid anti-oxidation solution. Plasma samples are extracted with 5 ml of methyl-t-butyl ether under alkaline conditions. The organic phase is evaporated to dryness and reconstituted with mobile phase. The quantitation range is 0.1 to 5.0 ng/ml (base) for both compounds. Mean ± S.D. recoveries for 4 concentrations within this range were 80.7 ± 4.3% for physostigmine and 84.1 ± 3.6% for eseroline. Interday and intraday (n - 6) coefficients of variation, respectively, for 4 concentrations in the 0.2 to 3.0 ng/ml range were 2.82 - 6.99% and 1.59 - 6.48% for physostigmine and 3.59 - 6.82% and 4.05 - 8.78% for eseroline. Bias for blind samples at 4 concentrations from 0.13 to 5.39 ng/ml ranged -12.5 to 3.46% for physostigmine and at 4 concentrations from 0.12 to 4.98 ng/ml ranged -16.6 to 43.1% for eseroline. Both compounds were found to be stable in prepared plasma when stored at -20°C for up to 4 months. The method was found to be reliable, fast, simple, accurate, precise, and capable of being used for routine analysis. Copyright © 1990 by Marcel Dekker, Inc.