Development of a liquid chromatography-ion mobility-mass spectrometry method for the screening of d-amino acid alterations in plasma

Probing chirality, one of the fundamental properties of all biological systems, remains analytically challenging and often requires specialized analytical setups or reagents, limited to few laboratories. This study aimed to develop an efficient liquid chromatography coupled to ion-mobility mass spectrometry (LC-IM-MS) method for the simultaneous analysis of all proteinogenic d-amino acids (d-AAs) in human plasma, using exclusively commercially available reagents. An indirect enantioseparation strategy was developed based on derivatization with (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE), with five stationary phases evaluated for diastereomer separation capability. A design of experiments approach was employed for further optimization, achieving baseline separation of all proteinogenic AA diastereomers within 20 min on a phenyl-based stationary phase. A straightforward sample preparation protocol was characterized, then the entire workflow was used for the assessment of d-AAs alterations across 60 plasma samples obtained from patients with chronic kidney disease (CKD, stages 4 and 5) and healthy controls. The analysis revealed increased levels of d-Pro, d-Arg, d-Ser, d-Asn, d-Gln, and d-Ala in CKD samples, highlighting the effectiveness of the developed analytical workflow to detect disease-related changes in plasmatic d-AAs.