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Comparison of 3-nitrophenylhydrazine, O-benzyl hydroxylamine, and 2-picolylamine derivatizations for analysis of short-chain fatty acids through liquid chromatography coupled with tandem mass spectrometry

  • Special Issue: Original Paper
  • Novel Analytical Technologies Contributing to Clinical and Pharmaceutical Research Fields
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Abstract

Short-chain fatty acids (SCFAs) are metabolites derived from gut microbiota and implicated in host homeostasis. Hence, the profiling SCFAs from biological samples plays an important role in revealing the interaction between gut microbiota and pathogens. Previous studies, liquid chromatography–tandem mass spectrometry (LC–MS/MS) combined with various derivatization strategies have been performed to obtain the SCFA profiles from biological samples. However, it is poor evidence to compare these derivatization regents and conditions. Thus, we present the evaluation of three major derivatization reagents, namely 3-nitrophenylhydrazine (3-NPH), O-benzylhydroxylamine (O-BHA), and 2-picolylamine (2-PA), for the analysis of eight SCFAs classified as C2–C5 isomers using LC–MS/MS. First, in a reversed-phase LC separation, 3-NPH showed good retention capacity. Although O-BHA derivatization showed higher sensitivity and good retention capacity than 2-PA, only 2-PA derivatization could successfully separate eight SCFAs. The matrix effects in human serum ranged 77.1–99.0% (RSD ≤ 3.4%, n = 6) for 3-NPH derivatives, 91.0–94.6% (RSD ≤ 5.4%, n = 6) for O-BHA derivatives, 81.6–99.5% (RSD ≤ 8.0%, n = 6) for 2-PA derivatives. These compared results showed each characteristic of 3-NPH, O-BHA, and 2-PA for SCFA derivatization based on LC–MS/MS approaches.

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Acknowledgements

This work was supported by JSPS KAKENHI Grant No. 22K06557.

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Authors and Affiliations

  1. College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga, 525-8577, Japan

    Ryosuke Nagatomo, Aoi Ichikawa, Haruki Kaneko & Koichi Inoue

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  1. Ryosuke Nagatomo
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  2. Aoi Ichikawa
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  3. Haruki Kaneko
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  4. Koichi Inoue
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Correspondence to Koichi Inoue.

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Nagatomo, R., Ichikawa, A., Kaneko, H. et al. Comparison of 3-nitrophenylhydrazine, O-benzyl hydroxylamine, and 2-picolylamine derivatizations for analysis of short-chain fatty acids through liquid chromatography coupled with tandem mass spectrometry. ANAL. SCI. 40, 843–851 (2024). https://doi.org/10.1007/s44211-023-00474-7

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