Determination of d -serine in human serum by LC-MS/MS using a triazole-bonded column after pre-column derivatization with ( S )-4-(3-isothiocyanatopyrrolidin-1-yl)-7- ( N , N -dimethylaminosulfonyl)-2,1,3-benzoxadiazole

Abstract

An LC-MS/MS-based method for determining d-serine (Ser), an endogenous co-agonist of the N-methyl-D-aspartate receptor, in human serum, was developed and validated using a triazole-bonded silica-packed column after pre-column fluorescence derivatization with a chiral labeling reagent, (S)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole. Enantiomeric separation of the d- and L-Ser derivatives occurred in the triazole-bonded column (R _s: 1.85) with CH₃CN/100 mM HCO₂NH₄ in H₂O (95.5:4.5) as the mobile phase with isocratic elution. The ln(capacity factor of d-Ser) in the van't Hoff plot gradually decreased with the inverse of temperature, suggesting enhanced hydrophilic interactions with the triazole-bonded stationary phase with increasing column temperature, owing to decrease in the partition coefficient to the mobile phase. Multiple reaction monitoring (m/z 457.10 > 409.00) by triple quadrupole mass spectrometry was used for quantifying d-Ser in human serum. The presence of d-Ser in the serum was confirmed by treatment with commercial d-amino acid oxidase. A linear calibration curve was constructed in the d-Ser concentration range of 0.5–5.0 μM (r ² = 0.999, n = 3) using d-homoserine as the internal standard. The precision and recovery values were adequate for quantification. The detection limit for d-Ser was 1.1 fmol/injection (signal-to-noise ratio = 3), owing to the high CH₃CN content in the mobile phase. The proposed LC-MS/MS method showed few fluctuations in the retention times of D- and L-Ser, and R _s was stable until the 40th injection of serum without column washing, and thus can be useful for d-Ser determination in human serum in clinical research.

Graphical Abstract

Multiple reaction monitoring chromatogram (left) and van't Hoff plot (right) of (S)-DBD-PyNCS-D- and L-serine on triazole-bonded column, obtained by LC-MS/MS

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