Dimethylcysteine (Dicys)/o-phthalaldehyde derivatization for chiral metabolite analyses: Cross-comparison of six chiral thiols

Abstract

Metabolomics profiling using liquid chromatography-mass spectrometry (LC-MS) has become an important tool in biomedical research. However, resolving enantiomers still repre-sents a significant challenge in the metabolomics study of complex samples. Here, we introduced N,N-dimethyl-L-cysteine (dimethylcysteine, DiCys), a chiral thiol, for the o-phthalaldehyde (OPA) derivatization of enantiomeric amine metabolites. We took interest in DiCys because of its potential for multiplex isotope-tagged quantification. Here, we characterized the usefulness of DiCys in reversed-phase LC-MS analyses of chiral metabolites, compared against five commonly used chiral thiols: N-acetyl-L-cysteine (NAC); N-acetyl-D-penicillamine (NAP); isobutyryl-L-cysteine (IBLC); N-(tert-butoxycarbonyl)-L-cysteine methyl ester (NBC); and N-(tert-butylthiocarbamoyl)-L-cysteine ethyl ester (BTCC). DiCys and IBLC showed the best overall performance in terms of chiral separation, fluorescence intensity, and ionization efficiency. For chiral separation of amino acids, DiCys/OPA also outperformed Marfey’s reagents: 1-fluoro-2-4-dinitrophenyl-5-L-valine amide (FDVA) and 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (FDAA). As proof of principle, we compared DiCys and IBLC for detecting chiral metabolites in aqueous extracts of rice. By LC–MS analyses, both methods detected twenty proteinogenic L-amino acids and seven D-amino acids (Ala, Arg, Lys, Phe, Ser, Tyr, and Val), but DiCys showed better analyte separation. We conclude that DiCys/OPA is an excellent amine-derivatization method for enantiomeric metabolite detection in LC-MS analyses.