title: Experimental design-based development of a rapid capillary electrophoresis method for determining impurities in the tetrapeptide H-Tyr-(D)Arg-Phe-Phe-NH2.
abstract: A capillary electrophoresis (CE) method has been developed and validated for separating the tetrapeptide H-Tyr-(D)Arg-Phe-Phe-NH2 and nine related substances. The method was developed using experimental design in a four-step procedure, in which eight variables were investigated in a total of 47 experiments. The preferred background electrolyte (BGE) consisted of 0.1M malonic acid at pH 2.5 with 7 mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin (2,6-DM-beta-CD). The separation of H-Tyr-(D)Arg-Phe-Phe-NH2 and the related substances was accomplished within 15 min, with a resolution greater than 1.5 between all peaks. The method was then investigated with respect to its selectivity, linearity, precision, detection limit (LOD) and quantitation limit (LOQ). In addition, a system suitability test was performed and response factors were determined, essentially following International Conference of Harmonization guidelines for the validation of analytical methods. LOD and LOQ for the related substance H-Arg-Phe-NH2 were found to be 0.3 and 0.8 microg/ml, respectively, at a target H-Tyr-(D)Arg-Phe-Phe-NH concentration of 1mg/ml. The method performed well with respect to all of the validation parameters.
authors.affiliation: Department of Analytical Chemistry, Stockholm University, Svante Arrhenius Vag 10-12, SE-106 91 Stockholm, Sweden.
authors.forename: Helena
authors.initials: H
authors.lastname: Brunnkvist
authors.forename: Bo
authors.initials: B
authors.lastname: Karlberg
authors.forename: Asa
authors.initials: A
authors.lastname: Astervik
authors.forename: Ingrid
authors.initials: I
authors.lastname: Granelli
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D009842:Oligopeptides / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C495156:H-Tyr-(D)Arg-Phe-Phe-NH2; D009842:Oligopeptides