Determination of Physostigmine in Plasma and Brain by HPLC*,**

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Abstract
A specific, reliable, and accurate high performance liquid chromatographic method is described for the determination of physostigmine in plasma and brain using carbaryl as an internal standard. Plasma and brain containing physostigmine were first precipitated with TCA, and then carbaryl was added. This was followed by chloroform extraction and then evaporation. The residue was reconstituted in the mobile phase. Physostigmine, its hydrolyzed product eseroline, and carbaryl were separated on a reversed-phase column eluted with mobile phase containing octanesulfonic acid with phosphate buffer in a methanol and water solution. The eluted compounds were detected at 245 nm, and physostigmine was quantified from the ratio of the area of physostigmine to carbaryl peaks. The chromatography was complete within 15 min. The dynamic range of quantitation of physostigmine was 0.05 µg to 0.5 µg/mL plasma or per gram of brain. Analytical recoveries varied from 95 to 107% over this range. Coefficient of variation ranged from 1.7 to 9.5%. This method was applied to study plasma and brain concentration in rats after 650 µg/kg intramuscular administration of physostigmine. The ratio of brain to plasma was found to be 0.48 and 1.97 at 15 and 30 min, respectively.