A rapid electrokinetic chromatography method using short-end injection for the enantioselective separation of tryptophan

Abstract

A rapid enantioselective methodology for the analysis of tryptophan was developed in this work by electrokinetic chromatography using short-end injection and an anionic cyclodextrin as chiral selector. No previous derivatization of tryptophan was necessary. The influence of different experimental variables on the enantiomeric separation was investigated. The use of a 100 mM formate buffer (pH 2.2) containing 1.25% sulfated-γ-cyclodextrin with an uncoated fused-silica capillary of 50 µm inner diameter with a total length of 48.5 cm (effective length of 8.5 cm), and an injection by applying a pressure of −50 mbar (short-end injection) for 4 s, enabled the enantiomeric separation of tryptophan within 2.5 min with a resolution of 7.4. As desirable, the enantiomeric impurity, D-tryptophan, was the first-migrating enantiomer. The analytical characteristics of the developed methodology were evaluated in terms of linearity, precision, accuracy, and limits of detection and quantification, showing its good performance to be applied to the analysis of tryptophan-based dietary supplements. A relative limit of detection of 0.1% was obtained for the enantiomeric impurity, D-tryptophan, in the presence of the L-enantiomer. Results showed that the developed methodology is an interesting alternative for the enantioselective analysis of tryptophan enabling the rapid quality control of dietary supplements.