Determination of muscimol in Amanita muscaria L. by high–performance thin–layer chromatography (HPTLC) method after decarboxylation

Abstract

Introduction The fly agaric well known as Amanita muscaria L. Ibotenic acid and muscimol are substances which mostly participate in psychotropic properties [1]. First symptoms of fly agaric intoxication apparent within 15 – 30 minutes after ingestion. Nausea, vomiting, diarrhea, vasodilation, sweating and salivation are the first signs of muscarinic poisoning [2]. A reliable analitycal method was developed for the identification of muscimol (MUS) and ibotenic acid (IBO). Materials and Methods Decarboxylation method was applied to convert ibotenic acid into muscimol [3]. The powder of lyophilizate herbal material (0.2g) were weighed onto aluminum plate and placed on the CAMAG TLC plate heater III for 1 hour. Temperature modes were selected 75C, 95C and 110C. After decarboxylation herbal substance was replaced into a 10 ml volumetric flask and extracted with 70% methanol (5ml) in an ultrasonic bath at room temperature for 10 min, with periodic shaking of solution. After 24 hours extract was centrifugated (4000 rpm) for 5min. We used high-performance thin-layer chromatography method (HPTLC) for qualitative and quantitative analysis of muscimol. Injection of muscimol standard solution was 5 μl and 10 μl for test solution. Chromatographic plates (HPTLC Silica gel 60 F254, 10x10 cm) coated with silica gel was used with 1- butanol:96% ethanol:acetic acid:water (75:25:5:7:5) as mobile phase, migration distance was over path 70 mm. The detection was performed after sprayed with a solution of ninhydrin, then plate was heated at 100C and examined with using CAMAG TLC Visualizer 2 in daylight. The obtained chromatograms of samples by the main spot correspond to the typical reference solution chromatogram with RF = 0.25. Calibration curve was done for quantitative determination. We counted of muscimol amount for determination of the most suitable temperature for decarboxylation. Results and discussion Three samples with different temperatures of heating w