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  • Title: Experimental design-based development of a rapid capillary electrophoresis method for determining impurities in the tetrapeptide H-Tyr-(D)Arg-Phe-Phe-NH2.
    Author: Brunnkvist H, Karlberg B, Astervik A, Granelli I.
    Journal: J Chromatogr B Analyt Technol Biomed Life Sci; 2004 Aug 05; 807(2):293-300. PubMed ID: 15203043.
    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.
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