title: Combination of ultracentrifugation and solid-phase extraction with subsequent chromatographic analysis of α-tocopherol in erythrocyte membranes.
abstract: A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid-phase extraction for the determination of α-tocopherol in human erythrocyte membranes by high-performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d-mannitol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and calcium chloride. The α-tocopherol was then extracted from the erythrocyte membranes by solid-phase extraction with n-hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α-tocopherol in real samples from patients. The combined ultracentrifugation and solid-phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid-liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes.
authors.affiliation: Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic; 3rd Internal Gerontometabolic Clinic, University Hospital, Hradec Králové, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Plíšek
authors.forename: Naďa
authors.initials: N
authors.lastname: Pospíchalová
authors.forename: Maria
authors.initials: M
authors.lastname: Khalikova
authors.forename: Jana
authors.initials: J
authors.lastname: Aufartová
authors.forename: Dagmar
authors.initials: D
authors.lastname: Solichová
authors.forename: Lenka Kujovská
authors.initials: LK
authors.lastname: Krčmová
authors.forename: Petr
authors.initials: P
authors.lastname: Solich
keywords: Alpha-tocopherol; Erythrocyte membranes; Red blood cells; Solid-phase extraction; Ultracentrifugation