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Simplification of FDLA Pre-Column Derivatization for LC/MS/MS Toward Separation and Detection of d,l-Amino Acids

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Abstract

Chiral chromatography is a sensitive technique used to separate l- and d-amino acids. Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-l-alaninamide, FDAA) has been used as a chiral derivatization reagent to determine amino acid stereochemistry. Replacement of alanylamide in this reagent with leucylamide resulted in the derivative 1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (FDLA), which proved a superior chiral derivatization reagent for the separation of d,l-amino acids due to the improved LC/MS measurement sensitivity. However, FDLA derivatization requires alkaline conditions, achieved with the use of NaHCO3, which necessitates an additional desalting step (e.g., liquid–liquid extraction) before quantitative analysis using LC/MS/MS. To establish a simplified LC/MS/MS measurement method, solid NaHCO3 was replaced with volatile triethylamine (TEA). The 30-min derivatization using FDLA under alkaline conditions with TEA at room temperature allowed for exclusion of the desalting step from the FDLA pre-column derivatization. Furthermore, using TEA, larger peak areas and more effective detection of hydrophilic amino acids could be achieved.

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Authors and Affiliations

  1. Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Sakado, 350-0295, Saitama, Japan

    Masaki Kobayashi, Yusuke Takano, Hiroki Takishima, Shintaro Sakaitani, Masaru Niitsu & Takemitsu Furuchi

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  1. Masaki Kobayashi
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  2. Yusuke Takano
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  3. Hiroki Takishima
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  4. Shintaro Sakaitani
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  5. Masaru Niitsu
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  6. Takemitsu Furuchi
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Correspondence to Takemitsu Furuchi.

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Kobayashi, M., Takano, Y., Takishima, H. et al. Simplification of FDLA Pre-Column Derivatization for LC/MS/MS Toward Separation and Detection of d,l-Amino Acids. Chromatographia 82, 705–708 (2019). https://doi.org/10.1007/s10337-018-3676-0

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  • DOI: https://doi.org/10.1007/s10337-018-3676-0

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