Quantification of alpha-amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection

• Type: Article in International Scientific Journal
• Date: 2015

Authors

Juliana Garcia (Author) Other; Vera M Costa (Author) Other; Paula Baptista (Author) Other; Maria de Lourdes Bastos (Author) FFUP; Felix Carvalho (Author) FFUP

Journal

Title: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

Vol. 997

Pages: 85-95

ISSN: 1570-0232

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 10 Citations

Scopus - 19 Citations

Scientific classification

FOS: Natural sciences > Chemical sciences

Other information

Authenticus ID: P-00G-CTD

DOI: 10.1016/j.jchromb.2015.06.001

Abstract (EN): alpha-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 alpha-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 x g, 4 degrees C, for 20 min. The high recovery found for alpha-amanitin (>= 96.8%) makes this procedure suitable for extracting alpha-amanitin from liver and kidney homogenates. The resulting supernatant was collected and injected into the HPLC. Mobile phase was composed by 20% methanol in 50 mM citric acid, and 0.46 mM octanessulfonic acid, adjusted to pH 5.5. The chromatographic runs took less than 22 min and no significant endogenous interferences were observed at the alpha-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 alpha-amanitin. In fact, the validated method was successfully applied to quantify alpha-amanitin in biological samples following intraperitoneal alpha-amanitin administration to rats. Moreover, human plasma was also used as matrix and the purposed method was adequate for detection of alpha-amanitin in that matrix. The results clearly indicate that the proposed method is suitable to investigate the pharmacokinetic and tissue distribution of alpha-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 alpha-amanitin toxicity.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 11