Microfluidic fast chiral separation of baclofen and phenylalanine enantiomers based on cyclodextrin-electrokinetic chromatography

• Published in: Microchemical journal 2021-01, Vol.160, p.105770, Article 105770
• Main Authors: Zeid, Abdallah M., Nasr, Jenny Jeehan M., Belal, Fathalla, Walash, Mohamed, Kaji, Noritada, Baba, Yoshinobu
• Format: Article
• Language: English
• Subjects: Biological samples; Chiral separation; Cyclodextrins; Fluorescence detection; Microchip electrophoresis
• ISSN: 0026-265X; 1095-9149
• DOI: 10.1016/j.microc.2020.105770

•A chiral MCE method for enantioseparation of baclofen and phenylalanine was developed.•Seven cyclodextrins were investigated to select the suitable chiral selector.•The microfluidic channels were dynamically coated with methylcellulose.•Field-enhanced stacking improved the sensitivity of the CD-MCE method.•The chiral CD-MCE was applied to analysis of dosage forms and biological samples. A green chiral lab-on-a-chip cyclodextrin-based microchip electrophoretic method was developed for enantioseparation of baclofen (BCN) and phenylalanine (Phe) enantiomers for the first time. All BCN and Phe enantiomers were offline labeled with 4-fluoro-7-nitrobenzofurazan (NBD-F) or fluorescein-5-isothiocyanate (FITC) fluorogenic reagents prior to microfluidic injection in a dynamically coated polymethylmethacrylate microchip. The enantioseparation was performed using borate buffer solution (50 mM; pH 9.5) containing a dynamic coating polymer (methylcellulose) and a chiral selector of cyclodextrin (CD) family as the background electrolyte. The combination of sieving properties of methylcellulose polymer and inclusion complex formation properties of the cyclodextrins rendered a novel green pseudo-stationary phase for the microfluidic chiral separation of the studied enantiomers in short separation time. Seven CD chiral selectors were investigated in our study to illustrate the proposed enantioseparation mechanism in the dynamically coated microfluidic channels. The results indicated that use of a modified cyclodextrin, heptakis-(2,6-di-O-methyl)-β-cyclodextrin (HDM-β-CD), resulted in extra interactions with the analytes of interest, leading to extra resolution with high number of theoretical plates. Therefore, HDM-β-CD was able to separate the NBD-labeled and FITC-labeled enantiomers of BCN and Phe within short analytical time. Sensitivity problem of microchip electrophoresis was overcome by application of field-enhanced injection stacking which improved sensitivity to a higher extent by online preconcentration of the labeled enantiomers in the microfluidic channels. The method was fully validated and successfully applied for the assay of BCN enantiomers and Phe enantiomers in their pure racemic mixtures. Moreover, the developed method was applied for precise analysis of BCN enantiomers in pharmaceutical formulations and in biological samples. The extraction recovery values of BCN enantiomers in biological samples were > 87.5% and %RSD