In Vitro Quantification of Hydrolysis-Induced Racemization of Amino Acid Enantiomers in Environmental Samples Using Deuterium Labeling and Electron-Impact Ionization Mass Spectrometry

d-Amino acids indicate aging, bacterial origin, and pathogenic properties of peptides in the environment, but the reliable assessment of d-enantiomers must account for a yet unknown formation during hydrolyses. Here, we introduce a method for the in vitro determination of the hydrolysis-induced racemization (HIR) of amino acids in environmental samples. It involves hydrolyses with hydro- and deuteriochloric acid (6 M, 12 h, 105 °C), desalting, and selective detection of chiral mass fragments of amino acid-N-pentafluoropropionyl derivatives. d-Amino acids formed in ²HCl incorporated deuterium into their C_α position. This resulted in a relative signal loss of the nondeuterated fragment compared with the ¹HCl hydrolysate. Mathematically evaluating the relative target signal intensities of both hydrolysates allowed the quantification of the proportion of d-amino acids formed during sample processing. Side-chain incorporations of deuterium were no limitations for this method as they could be estimated from that of the respective l-enantiomers. In soil and litter samples, between 0 (d-glutamic acid) and 85% (d-alloisoleucine) of the detected d-amino acids were formed upon hydrolysis (standard error, 5−11%). For a given amino acid, the HIR varied by a factor of 2−10 between samples, thereby confirming that HIR must be individually assessed for samples from different environments.