Derivatize, Racemize, and Analyze-an Easy and Simple Procedure for Chiral Amino Acid Standard Preparation for Enantioselective Metabolomics

A simple, controllable, and reproducible stereo-isomerization (racemization and epimerization) protocol for the preparation of scalemic alpha-amino acid mixtures from stereo-isomerically pure standards was developed. Simply derivatize your amino acids with a racemization tag that incorporates a urea bond on the N-terminus of the target amino acid and incubate at elevated temperatures up to 95 degrees C for defined time periods until the targeted D-amino acid levels are obtained. The racemization tags investigated were 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), aminophenyl-N-hydroxysuccinimidyl carbamate (AC), and 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APC). Employing this method, it was possible to create a ready-to-use, tailor-made chiral uniformly C-13 and N-15 labeled [U-(CN)-C-13-N-15]-amino acid standard with the desired D-amino acid percentage within minutes or hours without sample cleanup. A racemization time of 30 min at 95 degrees C will lead to a D-amino acid level of 1-5%, while 6 h at 95 degrees C provides 15-30% D-amino acids. Racemization occurs due to imine formation at the chiral carbon atom bound to the urea-linking group without decomposition of labile amino acids such as Asn, Gln, Trp, Cit, and theanine. For amino acids possessing two chiral centers such as DL-Ile or DL-Thr, only the epimerization of isomers with different stereochemistry at the second chiral center will produce all four possible isobaric enantiomers. All measurements were performed on the zwitterionic Chiralpak ZWIX(+) column using a dual hydro-organic flow gradient combined with HPLC-ESI-QTOF-MS analysis. This new racemization method solves the problem of (enantioselective) matrix effects and inaccurate results in LC-MS based enantioselective metabolomics and warrants full MS-compatibility.