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

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 1. Introduction Diketopiperazines (DKPs) are the smallest possible cyclic peptides composed of two -amino acids. They are abundant natural compounds produced by various bacteria like Streptomyces sp. [1], Pseudomonas aeruginosa [2], or Lactobacillus plantarum [3], fungi, e.g., Aspergillus flavus [4] or Alternaria alternata [5], and marine sponges like Dysidea herbacea [6]. Recently, the interest in this substance class has increased due to their immense bioactivities including antibacterial activity [7], antifungal function [3], cytotoxicity [4], phytotoxicity [5], and inhibition of plasminogen activator inhibitor-1 [8]. DKPs were shown to act as quorum sensing molecules; e.g., cyclo(L-Pro-L-Tyr), used in this study, was identified in culture supernatant of Pseudomonas aeruginosa and was identified as an activator of an N-acylhomoserine lactone biosensor [2]. Besides their widespread biosynthesis in nature, DKPs occur as chemical degradation products of, for example, amoxicillin, an aminopenicillin antibiotic [9], neuropeptide substance P [10], angiotensin converting enzyme inhibitor enalapril [11], or the sweetener aspartame with cyclo(L-Asp-L-Phe) as degradation product [12–17]. Amoxicillin and especially its degradation products can be detected in aquatic