Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine.
The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm x 25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio = 25. The linearity studied on spiked urine was satisfactory (r = 0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10-300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n = 10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.
Defendenti
C
C
Presidio Multizonale di Igiene e Prevenzione, Unità di Riferimento Regionale per la Micologia, Milano, Italy.
Bonacina
E
E
Mauroni
M
M
Gelosa
L
L
D000546:Amanitins / Q000737:chemistry / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D005554:Forensic Medicine; D006801:Humans; D009145:Mushroom Poisoning / Q000652:urine*; D015203:Reproducibility of Results
D000546:Amanitins