Stereoselective separation of 4-hydroxyproline by electrokinetic chromatography

Abstract

A chiral methodology was developed in this work enabling for the first time the separation of the four stereoisomers of the amino acid 4-hydroxyproline (4-Hyp) in the format of capillary electrophoresis (CE). After a screening of different neutral cyclodextrins (CDs) in the electrokinetic chromatography (EKC) mode, methyl-γ-CD was selected as chiral selector to stereoselectively separate 4-Hyp (previously derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl)) in a 75 mM phosphate buffer at pH 7.0. The effect of the concentration of the CD, the separation voltage, and the temperature on the chiral separation was investigated. A concentration of 10 mM for methyl-γ-CD, a voltage of 30 kV, and a temperature of 15 °C allowed the separation of the four stereoisomers of 4-Hyp in less than 21 min with resolutions between consecutive peaks of 1.5, 2.7, and 3.6. The injection of individual standard solutions of each stereoisomer enabled peak identification and the methodology was able to detect up to 0.1 % (1.3 × 10-11 mmol) of each stereoisomer. Analytical characteristics of the developed methodology were adequate to be applied to the analysis of nutricosmetic supplements. A good agreement was observed between the content determined for trans-4-l-Hyp and that indicated in the label for the product. No enantiomeric impurities were detected what shows the great potential of this method in the quality control of these products.