Development of a cyclodextrin-electrokinetic chromatography methodology for the separation of biopterin enantiomers

• First CD-EKC method for the chiral separation of biopterin • The use of S-b-CD enabled the separation in < 7.0 min with a resolution of 2.5 • Detection limits of around 0.1 μg/mL were obtained without sample preconcentration • The addition of DTT was needed to prevent the reduction of biopterin in urine • The method was applied to the chiral analysis of biopterin in urine. This work presents the development of an analytical methodology based on the use of cyclodextrin-electrokinetic chromatography to achieve the enantiomeric separation of biopterin, a biologically relevant molecule that plays a significant role as a cofactor for aromatic amino acid hydroxylases in mammals. A systematic study of the influence of different experimental and instrumental variables, such as the nature and pH of the buffer, the chiral selector and its concentration, the capillary length, the separation voltage, and the temperature, on the chiral separation was performed. Under optimized conditions (1.0 % sulfated-β-cyclodextrin in 100 mM formate buffer at pH 2.2), biopterin enantiomers were separated in less than 7.0 min with a resolution value of 2.5. The analytical characteristics of the developed method were evaluated in terms of linearity, precision, and limits of detection/quantification, in accordance with the International Council for Harmonization (ICH) guidelines. Limits of detection of 0.12 and 0.13 µg/mL for D- and L- biopterin were obtained. To demonstrate the potential applicability of the developed methodology, urine was selected as the biological matrix. Before analysis, urine samples were treated with dithiothreitol (DTT) to prevent oxidative-reductive changes of biopterin. Analysis of spiked urine samples allowed to evaluate mean recoveries for D- and L-enantiomers of 105 ± 5 % and 102 ± 8 %, respectively.