Development of a three-dimensional HPLC system for the determination of serine, threonine and allo-threonine enantiomers in the plasma of patients with chronic kidney disease

•A 3D-HPLC system was developed for the determination of chiral amino acids.•Enantiomers of serine, threonine and allo-threonine were determined.•All of the 3 target d-amino acids were successfully determined in human plasma.•The concentrations of 3 d-amino acids were increased along with the decreasing of renal function.•The d-forms of threonine and allo-threonine would be candidates as new biomarkers of CKD. A highly-selective three-dimensional high-performance liquid chromatographic (3D-HPLC) system was developed for the determination of serine (Ser), threonine (Thr) and allo-threonine (aThr) enantiomers in human plasma to screen the new biomarker of chronic kidney disease (CKD). d-Ser has been reported to be the candidate biomarker of CKD, however, multiple biomarkers are still required. Therefore, Ser analogs of hydroxy amino acids are the focus in the present study. For the sensitive analysis, the amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and detected by their fluorescence. The 3D-HPLC system consisted of a reversed-phase column (Singularity RP18, 1.0 × 250 mm), an anion-exchange column (Singularity AX, 1.0 × 150 mm) and a Pirkle-type chiral stationary phase (Singularity CSP-013S, 1.5 × 250 mm). The developed method was validated and applied to the human plasma samples obtained from 15 healthy volunteers and 165 CKD patients. The concentrations of the d-forms were 1.13–2.26 (Ser), 0.01–0.03 (Thr) and 0.04–0.10 μM (aThr) for the healthy volunteers and 0.95–19.0 (Ser), 0–0.57 (Thr) and 0.04–1.02 μM (aThr) for the CKD patients. The concentrations and the %d values of all the target d-amino acids were increased along with the decreasing of renal function and further investigation for clinical applications are expected.