title: Quantification of alpha-amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection.
abstract: α-Amanitin is a natural bicyclic octapeptide, from the family of amatoxins, present in the deadly mushroom species Amanita phalloides. The toxicological and clinical interests raised by this toxin, require highly sensitive, accurate and reproducible quantification methods for pharmacokinetic studies. In the present work, a high-performance liquid chromatographic (HPLC) method with in-line connected diode-array (DAD) and electrochemical (EC) detection was developed and validated to quantify α-amanitin in biological samples (namely liver and kidney). Sample pre-treatment consisted of a simple and unique deproteinization step with 5% perchloric acid followed by centrifugation at 16,000×g, 4°C, for 20min. The high recovery found for α-amanitin (≥96.8%) makes this procedure suitable for extracting α-amanitin from liver and kidney homogenates. The resulting supernatant was collected and injected into the HPLC. Mobile phase was composed by 20% methanol in 50mM citric acid, and 0.46mM octanessulfonic acid, adjusted to pH 5.5. The chromatographic runs took less than 22min and no significant endogenous interferences were observed at the α-amanitin retention time. Calibration curves were linear with regression coefficients higher than 0.994. The overall inter- and intra-assay precision did not exceed 15.3%. The present method has low interferences with simple and fast processing steps, being a suitable procedure to support in vivo toxicokinetic studies involving α-amanitin. In fact, the validated method was successfully applied to quantify α-amanitin in biological samples following intraperitoneal α-amanitin administration to rats. Moreover, human plasma was also used as matrix and the purposed method was adequate for detection of α-amanitin in that matrix. The results clearly indicate that the proposed method is suitable to investigate the pharmacokinetic and tissue distribution of α-amanitin. Additionally, the method will be very useful in the development of novel and potent antidotes against amatoxins poisoning and to improve the knowledge of α-amanitin toxicity.
authors.affiliation: REQUIMTE/Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua José Viterbo Ferreira n° 228, 4050-313 Porto, Portugal. Electronic address: jugarcia_18@hotmail.com.
authors.forename: Juliana
authors.initials: J
authors.lastname: Garcia
authors.affiliation: REQUIMTE/Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua José Viterbo Ferreira n° 228, 4050-313 Porto, Portugal.
authors.forename: Vera M
authors.initials: VM
authors.lastname: Costa
authors.affiliation: CIMO/School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-854 Bragança, Portugal.
authors.forename: Paula
authors.initials: P
authors.lastname: Baptista
authors.affiliation: REQUIMTE/Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua José Viterbo Ferreira n° 228, 4050-313 Porto, Portugal.
authors.forename: Maria de Lourdes
authors.initials: Mde L
authors.lastname: Bastos
authors.affiliation: REQUIMTE/Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua José Viterbo Ferreira n° 228, 4050-313 Porto, Portugal. Electronic address: felixdc@ff.up.pt.
authors.forename: Félix
authors.initials: F
authors.lastname: Carvalho
mesh_terms: D053959:Alpha-Amanitin / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D055664:Electrochemical Techniques / Q000379:methods*; D005260:Female; D006801:Humans; D007668:Kidney / Q000737:chemistry; D016014:Linear Models; D008099:Liver / Q000737:chemistry; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
chemical_list: D053959:Alpha-Amanitin
keywords: Diode-array; Electrochemical; High-performance liquid chromatographic; α-amanitin