Determination of physostigmine in plasma by high-performance liquid chromatography and fluorescence detection.
Physostigmine (PHY) is an anticholinergic drug used in the treatment of neuromuscular disorders and organophosphate poisoning. We described a sensitive, accurate, and reproducible method for PHY determination in biological materials. The method utilized a liquid/liquid, ion pair extraction, normal phase HPLC separation, and fluorometric quantitation at 240 nm excitation and 360 nm emission wavelength. We used neostigmine as a stabilizing agent to protect PHY from degradation and dimethylphysostigmine as an internal standard. The peak-height ratio vs concentration was linear over a working range from 0.50 to 25.0 ng/ml of PHY in plasma. Sensitivity of the method was 100 pg/ml of plasma which was the limit of quantitative detection under the experimental conditions used. Precision of the method was evaluated using plasma spiked with two concentrations of PHY: 1.0 and 10.0 ng/ml. Intra-day coefficient of variation (CV) ranged from 3.8 to 5.3%, and inter-day CV ranged from 1.8 to 3.6% for the two levels. The average recovery was 92%. We applied the method to examine the stability of PHY in plasma stored at -15 and -80 degrees C. The data indicated that PHY can be stored at either temperature for 9 weeks without undergoing significant alterations.
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Division of Toxicology, Letterman Army Institute of Research, Presidio of San Francisco, California 94129.
Ryabik
J R
JR
Ferraris
S
S
Wheeler
C R
CR
Korte
D W
DW
D002851:Chromatography, High Pressure Liquid; D006801:Humans; D010830:Physostigmine / Q000097:blood*; D012997:Solvents; D013050:Spectrometry, Fluorescence
D012997:Solvents; D010830:Physostigmine