Improved high performance liquid chromatographic determination of amanitins with electrochemical detection

Summary The determination of amanitins, the main toxins of the poisonous fungus Amanita Phalloides, through an improved HPLC method with electrochemical detection is presented. It uses an improved reverse phase separation on a wide pore butyl column and amperometric detection in oxidizing mode at + 0.6 V. Compared to the previously reported methods employing narrow pore C18 packings and UV detectors, the described method gives a much lower detection limit (40, 80 and 70 pg ca. for alpha-, beta- and gamma-amanitin, respectively), and a better chromatographic efficiency. Coupled with a sample preparation procedure using disposable cartridges packed with C18 and underivatized silica (recovery of alpha-amanitin =65 %, RSD 4.1 at 20 ng/ml), the method permits the quantitative determination of alphaamanitin in serum at low concentrations (10–20 ng/ml). A tentative immuno-extraction of urine samples, by means of a solid phase antiserum, bound to nylon nets, allowing the detection of few nanograms of toxin per milliliter is also briefly described.