Biaryl axially chiral derivatizing agent for simultaneous separation and sensitive detection of proteinogenic amino acid enantiomers using liquid chromatography–tandem mass spectrometry

Abstract

Novel sophisticated derivatizing agents for the efficient enantioselective separation and mass spectrometric detection of d- and l-amino acids have been developed. Two new axially chiral reagents derived from 6,6′-dimethyl-2,2′-biphenyldiamine were synthesized. Their chiral separation and detection abilities were evaluated by derivatizing proteinogenic amino acid standards in reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS). The resulting diastereomers derived from the developed derivatizing agents and amino acids could be completely separated, because of the effective chiral environment constructed by the axially chiral biphenyl moiety. After optimizing the reactive group, (R)-4-nitrophenyl N-[2′-(diethylamino)-6,6′-dimethyl-[1,1′-biphenyl]-2-yl]carbamate hydrochloride ((R)-BiAC) was found to be the best reagent for highly sensitive simultaneous d,l-amino acid analysis. Using (R)-BiAC, the complete chiral separation of all derivatized proteinogenic amino acids was achieved within 11.5 min with Rs greater than 1.9, except for certain allo-isomers. An exceptional feature of this reagent was its control of elution order, i.e., it afforded elution of the diastereomers derived from d-amino acids before their l-amino acid counterparts for all 19 proteinogenic amino acids. Sensitive detection was also achieved by introducing a dialkyl amino group and selectively cleaving it at the binding site between the reagent and amino acid. Attomole (amol) detection limits (signal-to-noise ratio = 3) were obtained for the tested d,l-amino acids, in the range 7.0–127 amol. As an example of application, the method was applied to food sample analysis, and detected several d-amino acids. Consequently, the developed method seems likely to facilitate simultaneous determination of enantiomers, including the tiny amounts of d-amino acids found in nature.