Table of Contents
Chromatography Research International
Volume 2013, Article ID 310269, 8 pages
Research Article

Separation of Cyclic Dipeptides (Diketopiperazines) from Their Corresponding Linear Dipeptides by RP-HPLC and Method Validation

Institute of Process Engineering in Life Sciences, Section II: Technical Biology, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, Germany

Received 1 September 2012; Accepted 18 November 2012

Academic Editor: Toyohide Takeuchi

Copyright © 2013 Mareike Perzborn et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Simple, rapid, sensitive, precise, and accurate methods for detection and separation of seven diketopiperazines (DKPs), cyclo(Gly-Gly), cyclo(DL-Ala-DL-Ala), cyclo(L-Asp-L-Phe), cyclo(L-Asp-L-Asp), cyclo(Gly-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Arg-L-Arg), from their corresponding linear dipeptides and related amino acids L-Phe and L-Tyr by reversed-phase high-performance liquid chromatography (RP-HPLC) were established. Moreover, for the racemic DKP cyclo(DL-Ala-DL-Ala) and dipeptide DL-Ala-DL-Ala, separation of the diastereomers was achieved. All methods can be performed within 15 min. For all DKPs, dipeptides, and amino acids, linear ranges with correlation coefficients greater than 0.998 were determined. Lowest limits of detection were found to be between 0.05 and 10 nmol per 10 μL injection, depending on the substance. For all tested substances intrarun and interrun precision ranged from 0.5 to 4.7% and 0.7 to 9.9% relative standard deviation, and accuracy was between −4.2 and 8.1% relative error. Short-term and freeze-thaw stabilities were 93% or greater for all substances. Recovery rate after heat treatment was determined to be at least 97%. These methods will be useful for quantitative determination of DKPs and their potential biodegradation products: dipeptides and amino acids