Leveraging Multi-Mode Microextraction and Liquid Chromatography Stationary Phases for Quantitative Analysis of Neurotoxin β-N-methylamino-L-alanine and Other Non-proteinogenic Amino Acids

Author(s): Ronald V. Emmons^1,2, Endri Karaj³, Erasmus Cudjoe⁴, David S. Bell⁵, L.M. Viranga Tillekeratne³, Emanuela Gionfriddo^1,2,6
1. Department of Chemistry and Biochemistry, The University of Toledo, Toledo, OH 43606, United States, 2. Dr. Nina McClelland Laboratory for Water Chemistry and Environmental Analysis, The University of Toledo, 3. Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, 4. Perkin Elmer Inc., Woodbridge, ON, L4L 8H1, Canada, 5. Restek Corporation, 6. School of Green Chemistry and Engineering, The University of Toledo

Published By: Journal of Chromatography A

Issue: Volume 1685

Year of Publication: 2022

Link: https://doi.org/10.1016/j.chroma.2022.463636

Abstract: Effective quantitative analysis of BMAA (β-N-methylamino-L-alanine) and its isomers without the need for derivatization has always been an analytical challenge due to their poor retention and separation on various liquid chromatography stationary phases. Previous studies that utilized conventional hydrophilic interaction chromatography (HILIC) demonstrate false negatives compared to reverse-phase workflows with derivatization. This work evaluates the chromatographic behavior of BMAA and its isomers, in their underivatized forms, on selected stationary phases, in particular fluorophenyl-based columns, to attain effective retention and separation. Detection and quantification were achieved with an ion-trap mass spectrometer. Extraction and preconcentration were achieved via solid phase microextraction (SPME) by assessing the effectiveness of multiple extraction phases, including hydrophilic-lipophilic balanced (HLB) and mixed-mode (MM). A MM extraction phase consisting of C₈ and benzene sulfonic acid moieties provided ideal extraction performance for BMAA and its isomers (2,4-diaminobutyric acid, DABA; N-(2-aminoethyl) glycine, AEG). Chromatographic separation was achieved within 8 min on a fluorophenyl stationary phase, ensuring high throughput without derivatization, and showing exceptional improvement from conventional HILIC methods. Limits of quantification in water for BMAA and AEG were 2.5 µg L⁻¹ and DABA was 5 µg L⁻¹, with linear dynamic ranges from 2.5 µg L⁻¹ - 200 µg L⁻¹ for BMAA and AEG and 5 µg L⁻¹ - 200 µg L⁻¹ for DABA.