Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine |
| |
| Affiliation: | 1. Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047;2. Department of Formulation Sciences, MedImmune LLC, Gaithersburg, Maryland 20878;3. Department of Chemistry, University of Kansas, 1251 Wescoe Hall Dr, Lawrence, Kansas 66045;1. Divisions of Hepatology and Critical Care Medicine, University of Alberta, Edmonton, Alberta, Canada;2. Section of Hepatology, Hume-Lee Transplant Center of Virginia Commonwealth University, Richmond, VA, USA;1. Faculty of Pharmaceutical Sciences, Sojo University, Japan;2. Graduate School of Pharmaceutical Sciences, Kyushu University, Japan;1. Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047;2. Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047;3. Department of Chemistry and R.N. Adams Institute of Bioanalytical Chemistry, University of Kansas, Lawrence, Kansas 66045 |
| |
| Abstract: | 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×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. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
