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

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 NaHCO 3 , 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 NaHCO 3 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.