title: Development and validation of a single HPLC method for determination of α-tocopherol in cell culture and in human or mouse biological samples.
abstract: A straightforward and common analytical method for α-tocopherol (αT) determination in various biological samples, including plasma, red blood cells (RBC), tissues and cultured cell lines, was developed and validated, using a reverse phase-chromatographic method (RP-HPLC). Even though many chromatographic methods for αT determination have been reported, most of them require readjustment when applied to different types of samples. Thus, an effective and simple method for αT determination in different biological matrices is still necessary, specifically for translational research. This method was applied using a C18 column (250 × 4.6 mm, 5 µm particle size) under isocratic elution with MeOH:ACN:H2 O (90:9:1 v/v/v) at a flow rate of 1 mL/min and detected using photodiode array at 293 nm. Linearity (r >0.9997) was observed for standard calibration with inter- and intraday variation of standard <4%. Lower limits of detection and quantification for αT in this assay were 0.091 and 0.305 µg/mL respectively. Validation proved the method to be selective, linear, accurate and precise. The method was successfully applied in great variety of biological samples, that is, human and mouse plasma, RBCs, murine tissues and human/mouse/rat cultured cell lines. More importantly, a single protocol of extraction and detection can be applied, making this method very convenient for standardization of different types of samples.
authors.affiliation: Departamento de Morfología y Biología Celular, Facultad de Medicina y Ciencias de la Salud, University of Oviedo, Oviedo, Spain.
authors.forename: Henar M
authors.initials: HM
authors.lastname: Cimadevilla
authors.affiliation: Departamento de Morfología y Biología Celular, Facultad de Medicina y Ciencias de la Salud, University of Oviedo, Oviedo, Spain.|Instituto Universitario Oncológico del Principado de Asturias, Oviedo, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Hevia
authors.affiliation: Departamento de Morfología y Biología Celular, Facultad de Medicina y Ciencias de la Salud, University of Oviedo, Oviedo, Spain.|Instituto Universitario Oncológico del Principado de Asturias, Oviedo, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Miar
authors.affiliation: Departamento de Morfología y Biología Celular, Facultad de Medicina y Ciencias de la Salud, University of Oviedo, Oviedo, Spain.|Instituto Universitario Oncológico del Principado de Asturias, Oviedo, Spain.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Mayo
authors.affiliation: Instituto Universitario Oncológico del Principado de Asturias, Oviedo, Spain.|Departamento de Biología Funcional, Facultad de Medicina y Ciencias de la Salud, University of Oviedo, Oviedo, Spain.
authors.forename: Felipe
authors.initials: F
authors.lastname: Lombo
authors.affiliation: Departamento de Morfología y Biología Celular, Facultad de Medicina y Ciencias de la Salud, University of Oviedo, Oviedo, Spain.|Instituto Universitario Oncológico del Principado de Asturias, Oviedo, Spain.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Sainz
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes; D006801:Humans; D016014:Linear Models; D008099:Liver / Q000737:chemistry; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D024502:alpha-Tocopherol / Q000032:analysis / Q000097:blood* / Q000737:chemistry
chemical_list: D024502:alpha-Tocopherol
keywords: HPLC; cell; plasma; tissue; α-tocopherol