Quantitative analysis of D/L-serine and D/L-proline in serum by isotope-coded derivatization using an original chiral resolution labeling reagent

The isotope-coded derivatization method was developed for the accurate quantification of D/L-serine and D/L-proline in serum by liquid chromatography-single quadrupole mass spectrometry (LC–MS), using our original chiral resolution labeling reagent labeled with the stable isotope 1-fluoro-2,4-dinitrophenyl-5-D-leucine-¹³C₆-N,N-dimethylethylenediamineamide (¹³C₆-D-FDLDA). These D-amino acids are potential biomarkers for Alzheimer’s disease (AD). The method enables straightforward separation and detection of D/L-serine and D/L-proline using an octadecyl (C₁₈) column after liquid–liquid extraction and derivatization. Moreover, the labeled samples remained stable for at least 1 month when stored at 4°C. The concentrations of D-serine (2.07 ± 0.07 μmol/L) and D-proline (1.50 ± 0.04 μmol/L) in the serum of AD patients were higher than those in non-AD (D-serine: 1.80 ± 0.06 μmol/L; D-proline: 0.43 ± 0.03 μmol/L), consistent with findings from previous studies. Extraction recovery rates for each amino acid were within ±15 %, and no carryover was detected immediately after serum sample analysis. Furthermore, this method enables accurate analysis of D/L-serine and D/L-proline in serum with RSD values below 10 % for intra-day and inter-day reproducibility. This method is promising for enabling early and minimally invasive diagnosis of patients with AD.