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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

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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.

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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Acknowledgments

The authors gratefully acknowledge Nacalai Tesque Co. Ltd. for valuable advice on the use of the triazole-bonded column, and Shimadzu Corporation for technical advice on the operation of LCMS-8040. The authors also thank Miss Megumi Iwasaki, Toho University, for her technical assistance. This study was partially supported by a Grant-in-Aid for Scientific Research (C) (no. 25460224) from the Japan Society for the Promotion of Science from the Ministry of Education, Culture, Sports, Science, and Technology. The authors declare no conflicts of interest.

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Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba, 274-8510, Japan

    Tatsuya Sakamoto, Ryousuke Kuwabara, Shuhei Takahashi, Mayu Onozato, Hideaki Ichiba, Hideaki Iizuka & Takeshi Fukushima

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  1. Tatsuya Sakamoto
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  2. Ryousuke Kuwabara
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  3. Shuhei Takahashi
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Correspondence to Takeshi Fukushima.

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Sakamoto, T., Kuwabara, R., Takahashi, S. et al. 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. Anal Bioanal Chem 408, 517–526 (2016). https://doi.org/10.1007/s00216-015-9119-y

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