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- 13 C 6 - N , N -dimethylethylenediamineamide ( 13 C 6 - 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 (C 18 ) 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. • Isotope-coded derivatization enables accurate analysis of D/L-serine and D/L-proline in serum. • DL-Amino acids labeled with our labeling reagent show high resolution and sensitivity. • The sample labeled with our labeling reagent remained stable for at least 1 month at 4°C. • This method has high precision with intra- and inter-day reproducibility within 10 %. • The %D of serine and proline in serum was significantly higher in AD patient than in non-AD.