| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 104 === The study of complex metabolites in biological samples is a rapidly advancing field. Tremendous progress has been made using advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques to analyze human metabolomes. It is now believed that human serum alone contains over 10,000 quantifiable metabolites. However, there is no universal LC-MS/MS condition that suits all metabolites. Instead of constantly tuning LC-MS conditions for different metabolites, a better approach is to derivatize metabolites to give them more desirable properties such as better LC separation efficiency, enhanced ionization efficiency, and favorable MS/MS fragmentation patterns.
In this study we focus on identifying optimal derivatization methods for amine-containing metabolites in LC-MS/MS analysis coupled with fluorescence detection. These metabolites may include amino acids, their derivatives, and peptides, which may function as hormones, neurotransmitters, and other signaling molecules in the body. We surveyed a wide variety of amine-derivatization regents and narrowed the candidate list down to five: o-phthlaldehyde (OPA), Dansyl-Cl, Dabsyl-Cl, Fmoc-Cl and Marfey’s reagent. We compared them in terms of absorbance intensity, product hydrophobicity, fluorescence intensity and pH dependence, separation efficiency in reversed-phase LC, ionization efficiency and salt dependence, as well as MS/MS fragmentation energy and fingerprint. To our knowledge such detailed comparisons of amine derivatization methods have never been carried out before.
In this study we compared three general aqueous mobile phase compositions: 0.1% FA (pH 2.6), 2 mM AA (pH 5), 2 mM ABC (pH 8). Under respective optimal eluent conditions, we have observed these general trend in terms of absorbance intensity (Dansyl > Marfey > Fmoc > Dabsyl > OPA), fluorescence intensity (Fmoc > OPA > Dansyl), hydrophobicity (Dabsyl > Fmoc > Dansyl ≈ Marfey ≈ OPA), and ionization efficiency (Dansyl ≈ Dabsyl > OPA ≈ Fmoc > Marfey). Fmoc and Dansyl exhibit characteristic product ions with fixed m/z in collision-induced dissociation cell, while OPA and Marfey show characteristic fixed mass loss in fragment product ions. Dabsyl fragments at many positions to create a complex MS/MS spectrum.
After extensive comparisons, we found that Dansyl shows the greatest potential for a universal derivatization method for metabolomics studies, especially for quantitation by fluorescence and multiple-reaction monitoring. Fmoc is a similarly useful reagent and has the advantage of low collision energy. OPA is a versatile fluorogenic reagent and its chemistry can be fine-tuned using different thiol molecules, which is worth further investigating and optimizing. Marfey’s reagent is useful for the chromatographic separation of enantiomers due to its chiral nature. Dabsyl is very difficult to fragment in MS/MS experiemnts, which may be a strength or a weakness depending on analytical goals. The performance comparisons between different reagents derived from this study can serve as a guide for designing better metabolomics experiments under different contexts.
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