A Radiometric High-Performance Liquid Chromatographic Assay for Physostigmine
Abstract
This method was developed to determine the pharmacokinetic parameters of 3H-physostigmine (3H-Phy) from serial plasma samples of small animals. A 200 ul plasma sample, mixed with 50 uls of aqueous neostigmine (10 mg/ml) to inhibit in vitro metabolism, was membrane filtered (10,000 mol wt limit). Filtrate (150 uls) was injected onto an HPLC instrument containing a 100 ul sample loop, C18 column and flow-through scintillation counter. Flow rates of mobile phase (5.0 mM 1-octanesulfonic acid, 5.0 mM sodium phosphate, 0.175 mM acetic acid aqueous buffer:methanol (60:40)) and scintillation fluid were 1.2 and 4.0 ml/min, respectively. Areas under the curves of cpm versus time for 0.1, 0.5, 1.0 and 5. 0 mg 3H-Phy/ml were used to create weighted standard curves by reciprocal of variance (n = 6). Physostigmine was totally resolved from eseroline. Sensitivity for physostigmine was 0.05 ng/ml; extraction efficiency + SD was 99.6 + 4.4% (n = 6). Correlation coefficients representing standard curve linearity ranged from 0.9697 to 0.9999 (n = 6). Within-day and between-day coefficients of variation (n = 6). Within-day and between-day coefficients of variation (n = 6) for 0.2, 0.75, 1.5 and 2.5 ng 3H-Phy/ml ranged from 0.7 to 20% and 16 to 32%, respectively. The sensitivity, linearity and precision of this method suggest that it should be able to accurately measure 3H-Phy concentrations in small plasma volumes to obtain pharmacokinetic data for small animals.
Document Details
- Document Type
- Technical Report
- Publication Date
- Nov 01, 1988
- Accession Number
- ADA203677
Entities
People
- Brian J. Lukey
- Claire N. Lieske
- Connie R. Clark
- Michael P. Mccluskey
Organizations
- United States Army Medical Research Institute of Chemical Defense