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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: 1.85) with CHCN/100 mM HCONH in HO (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 = 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 CHCN 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 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. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]