Development of an electrokinetic chromatography method for the rapid enantiomeric determination of 5-hydroxytryptophan. Application to the analysis of dietary supplements

Abstract

5-Hydroxytryptophan is a chiral non-proteinogenic amino acid that plays a key role in the metabolic and neurological balance of an organism, as it is the precursor of serotonin. Dietary 5-hydroxytryptophan supplementation based on Griffonia simplicifolia seed extracts is widely used to enhance positive emotion recognition. Since the L-form of this amino acid exhibits the desired biological activity, and legal regulations do not allow the presence of the D enantiomer in dietary supplements, their quality control requires the development of chiral methodologies. This work describes the development of a rapid electrokinetic chromatography method for the enantiomeric determination of 5-hydroxytryptophan and its application to the analysis of a dietary supplement. The developed strategy was based on the use of 1.75% (w/v) sulfated-γ-cyclodextrin in 100 mM formate buffer (pH 2.2) as a separation medium, a short-end sample injection, and an uncoated fused-silica capillary with an effective length of 8.5 cm (total length of 48.5 cm). Under the optimized conditions, the enantiomeric separation of 5-hydroxytryptophan was achieved in less than 3 min with a resolution of 4.6. The analytical characteristics of the developed methodology were evaluated, showing a good performance in terms of linearity (R > 0.985), precision (RSD <6.0% for migration times and <8.6% for peak areas), accuracy (90 ± 4%) and LOQs (0.42 and 048 mg L⁻¹ for D and L-5-hydroxytryptophan, respectively), and it was successfully applied to the quality control of a Griffonia simplicifolia dietary supplement rich in L-5-hydroxytryptophan.