Simple liquid chromatographic method for the determination of physostigmine and its metabolite eseroline in rat plasma: application to a pharmacokinetic study

Physostigmine, an anticholinergic drug, and its metabolite eseroline can be quantitated by high-performance liquid chromatography (HPLC) with photodiode-array detection. After addition of the structurally related internal standard (−)- N-methylphysostigmine, rat plasma samples were extracted and cleaned using a Varian Bond Elut C 18 column. The methanol–ammonia (98:2) eluate was evaporated to dryness and reconstituted with 0.01 M sodium dihydrogenphosphate (pH 3). Physostigmine and eseroline were separated on an Alltech Ultrasphere Silica column (250×4.6 mm I.D.; particle size 5 μm) at a flow-rate of 1 ml/min, with a mobile phase of 0.01 M sodium dihydrogenphosphate (pH 3)–acetonitrile (85:15). The limits of detection were 10 and 25 ng/ml for physostigmine and eseroline, respectively; the signal-to-noise ratio for this concentration was approximately 3:1. Spiked rat plasma containing 0.1–2.5 μg/ml of physostigmine and eseroline gives good linearity. The average percentage recovery from five spiked plasma samples was 88.0±2.9 and 61.1±5.6% for physostigmine and eseroline, respectively. Within the concentration range 0.1–2.5 μg/ml, the within-day precision was 1.9–8.3 and 3.0–7.7% for physostigmine and eseroline, respectively, and the between-day precision was 4.1–9.3 and 3.7–11% for physostigmine and eseroline, respectively. The method is rapid, simple and reliable, thus it is suitable for pharmacokinetic studies in the rat.