Simultaneous enantiomeric separation of tryptophan-related metabolites by cyclodextrin-electrokinetic chromatography. Application to the chiral analysis of kynurenine and tryptophan in urine samples

Tryptophan (Trp) is an essential chiral amino acid that plays a fundamental role in numerous physiological and neurological processes. It serves as a key compound for the synthesis of biologically significant metabolites, including kynurenine (Kyn), 5-hydroxytryptophan (5-HTP), and serotonin (5-HT); each of which contributes to distinct yet interconnected biochemical functions. Except 5-HT, the other two tryptophan-related metabolites are chiral molecules. In this study, the simultaneous separation of 5-HT from Trp, Kyn, and 5-HTP enantiomers, was achieved for the first time using cyclodextrin-electrokinetic chromatography (CD-EKC) with sulfated-γ-CD as the chiral selector. Optimization of the chiral selector concentration, together with fine-tuning of the temperature and separation voltage, enabled the simultaneous enantiomeric resolution of all target chiral metabolites and 5-HT, achieving resolution values higher than 3.7 in less than 20 min. Since the kynurenine pathway represents the main catabolic route of Trp metabolism, the developed methodology was applied to the determination of Trp and Kyn at their physiological levels in urine samples. Prior to its application, the analytical performance of the method was evaluated, demonstrating good suitability for the determination of both metabolites with LOQs of 0.29 μg/mL for L-Kyn and 1.45 μg/mL for L-Trp. Urine samples were submitted to a freeze-thaw desalting step followed by solid-phase extraction before CD-EKC analysis. • Tryptophan-related metabolites were simultaneously enantioseparated by CD-EKC • Kynurenine, tryptophan, and 5-hydroxytryptophan enantiomers separated from serotonin • Sulfated-γ-CD provided resolution values between adjacent peaks higher than 3.7 • LODs of 0.09 μg/mL for L-kynurenine and 0.43 μg/mL for L-tryptophan were obtained • L-kynurenine and L-tryptophan quantified in human urine after SPE treatment