title: Design of experiments for amino acid extraction from tobacco leaves and their subsequent determination by capillary zone electrophoresis.
abstract: In this study, we optimized a method for the determination of free amino acids in Nicotiana tabacum leaves. Capillary electrophoresis with contactless conductivity detector was used for the separation of 20 proteinogenic amino acids in acidic background electrolyte. Subsequently, the conditions of extraction with HCl were optimized for the highest extraction yield of the amino acids because sample treatment of plant materials brings some specific challenges. Central composite face-centered design with fractional factorial design was used in order to evaluate the significance of selected factors (HCl volume, HCl concentration, sonication, shaking) on the extraction process. In addition, the composite design helped us to find the optimal values for each factor using the response surface method. The limits of detection and limits of quantification for the 20 proteinogenic amino acids were found to be in the order of 10-5and 10-4mol l-1, respectively. Addition of acetonitrile to the sample was tested as a method commonly used to decrease limits of detection. Ambiguous results of this experiment pointed out some features of plant extract samples, which often required specific approaches. Suitability of the method for metabolomic studies was tested by analysis of a real sample, in which all amino acids, except for L-methionine and L-cysteine, were successfully detected. The optimized extraction process together with the capillary electrophoresis method can be used for the determination of proteinogenic amino acids in plant materials. The resulting inexpensive, simple, and robust method is well suited for various metabolomic studies in plants. As such, the method represents a valuable tool for research and practical application in the fields of biology, biochemistry, and agriculture.
authors.affiliation: Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 00, Prague 2, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Hodek
authors.affiliation: Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 00, Prague 2, Czech Republic. tomas.krizek@natur.cuni.cz.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Křížek
authors.affiliation: Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 00, Prague 2, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Coufal
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 00, Prague 2, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Ryšlavá
mesh_terms: D000596:Amino Acids / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D008962:Models, Theoretical; D018515:Plant Leaves / Q000737:chemistry*; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D000596:Amino Acids
keywords: Amino acids; Capillary electrophoresis; Central composite design; Factorial design; Response surface method; Tobacco