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

Abstract

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 R2 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