Solid phase extraction for an improved assay of physostigmine in biological fluids

Abstract

A simple, selective and very sensitive assay is described for the quantification of physostigmine in blood, plasma and urine. The most appropriate solid phase column was selected after a systematic investigation of nine types of phase. The conditions for solid phase extraction were optimized using [3H]physostigmine so that the overall recoveries were greater than 90%. Physostigmine was retained on alkaline treated cyanopropyl columns and eluted into the minimum volume of methanol, obviating the need for an evaporation step. Extracted samples were quantified by HPLC with a three electrode coulometric detection system. The limit of detection was 50 pg/mL for a 0.5 mL plasma sample. The precision (CV) for 0.5 mL plasma samples containing 50 pg was 8.1%. Application of the method to plasma, blood and urine samples is presented.

The binding of physostigmine to human serum albumin

“…The Michaelis constant for the degradation of physostigmine in human plasma is 76 nM (Hurst & Whelpton 1989) and a dissociation constant of 12.6 nM is not inconsistent with this. The hydrolysis of physostigmine by cholinesterase is complex, the rate of reaction being increased by the presence of substrate (Berry 1971).…”

“…2) showed only slight degradation of physostigmine during the course of the experiment (from 98% to 96.4%). The major radioactive impurity in the original chromatographed with the same retention volume as ['Hlwater (Hurst & Whelpton 1989) whereas there are clearly additional peaks with retention times using only 4 initial concentrations. equivalent to the hydrolysis product eseroline and its oxidation product, rubreserine.…”

“…The purity of the physostigmine * Correspondence and present address: R. Whelpton, Department of Pharmacology, Faculty of Basic Medical Sciences, Queen Mary and Westfield College, Mile End Road, London El 4NS, UK. before and after dialysis was determined by chromatographing the samples on a cyanopropyl HPLC column and collecting fractions for scintillation counting (Hurst & Whelpton 1989).…”

Measurement of terbutaline and salbutamol in plasma by high performance liquid chromatography with fluorescence detection

“…Seven types of Bond-Elut phases (C2, CX, C18, CN, DioL, Si and SCX; Jones) were evaluated. The columns were prepared by sequentially washing with methanol (2 x 1 mL), water (2 x 1 mL) and 1 mL dipotassium orthophosphate (0.1 mol/L, pH 9.2) using the Vac-Elut manifold (Hurst and Whelpton, 1989). Solutions containing terbutaline, salbutamol and atenolol in blood blank plasma or water (1 mL, 10 mg/L) were applied.…”

Biologically active components of Physostigma venenosum

“…Denaturation with acids or more than 0.1 ml of acetonitrile reduced the extraction recoveries of the analytes. In another study conducted by Hurst and Whelpton [51], an alkaline treated cyanopropyl column was selected after a systematic investigation of nine types of phase. Physostigmine was retained on the cyanopropyl columns and eluted into the minimum volume of methanol.…”