Related Experiment Video
Updated: Jan 7, 2026
09:18
Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics
Published on: April 17, 2017
10.2K
Distinguishing 4- vs 5-Hydroxy-N,N-Dimethyltryptamine (Psilocin vs Bufotenine) Using Hydrogen-Deuterium Back-Exchange
Michael W Christopher1, Boone M Prentice1, Timothy J Garrett1,2
1Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States.
Journal of the American Society for Mass Spectrometry
|December 30, 2025
Summary
This study introduces a novel method using hydrogen-deuterium exchange (HDX) to differentiate metabolite isomers based on their unique pKa values. This technique accurately distinguishes psilocin and bufotenine, crucial for analyzing complex biological samples.
Area of Science:
- Analytical Chemistry
- Metabolomics
- Physical Chemistry
Background:
- Distinguishing metabolite isomers typically requires comparative data (e.g., retention times, ion mobility, product ion abundances), necessitating reference standards.
- An ideal isomer differentiation method should utilize absolute physicochemical properties, independent of instrument specifics or external data.
Purpose of the Study:
- To develop and validate a method for resolving positional isomers of hydroxy-N,N-dimethyltryptamine (psilocin and bufotenine) using their distinct pKa values.
- To establish a technique that relies on absolute physicochemical properties rather than relative measurements or reference standards.
Main Methods:
- Employing hydrogen-deuterium exchange (HDX) to selectively exchange hydrogen atoms with deuterium.
- Utilizing liquid chromatography (LC) for back-exchange of the amine deuterium.
- Monitoring kinetic deuterium exchange rates of the aromatic hydroxy group via high-resolution mass spectrometry (HRMS).
Main Results:
- The HDX kinetics effectively differentiated between 4- and 5-hydroxy positional isomers.
- 4-hydroxy-N,N-dimethyltryptamine (psilocin) showed significant deuterium exchange within hours, while 5-hydroxy-N,N-dimethyltryptamine (bufotenine) did not.
Conclusions:
- Leveraging pKa differences through HDX kinetics provides a robust method for isomer differentiation.
- This approach offers a universal strategy for characterizing unknown structural isomers without reliance on external references or specific instrumentation.
Related Concept Videos
01:20¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)
1.6K
When proton-coupled carbon-13 spectra are simplified by a broadband proton decoupling technique, structural information about the coupled protons is lost. Distortionless enhancement by polarization transfer (DEPT) is a technique that provides information on the number of hydrogens attached to each carbon in a molecule. While the DEPT experiment utilizes complex pulse sequences, the pulse delay and flip angle are specifically manipulated. The resulting signals have different phases depending on...
1.6K
00:48¹H NMR of Labile Protons: Deuterium (²H) Substitution
1.3K
This lesson illustrates the role of deuterium substitution in simplifying the NMR spectrum of compounds comprising labile protons. One method employed is the use of deuterium. Amongst the three isotopes of hydrogen, deuterium (2H) has a nucleus composed of one proton and one neutron. When the D2O solvent is added to a pure dry ethanol solution, its labile proton is substituted with deuterium.
1.3K
01:13¹H NMR: Complex Splitting
1.8K
A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied...
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied...
1.8K
01:03¹H NMR Chemical Shift Equivalence: Homotopic and Heterotopic Protons
4.0K
Protons in identical electronic environments within a molecule are chemically equivalent and have the same chemical shift. The replacement test is a useful tool to identify chemical equivalence and predict NMR spectra. A substituent replaces each of the protons being examined and the resulting molecules are compared. If the same molecule is obtained, the protons are equivalent or homotopic. Replacement of any hydrogens in ethane by chlorine yields chloroethane because all six protons are...
4.0K
00:58¹H NMR Chemical Shift Equivalence: Enantiotopic and Diastereotopic Protons
3.1K
Replacing each alpha-hydrogen in chloroethane by bromine (or a different functional group) yields a pair of enantiomers. Such protons are called prochiral or enantiotopic and are related by a mirror plane. Enantiotopic protons are chemically equivalent in an achiral environment. Because most proton NMR spectra are recorded using achiral solvents, enantiotopic hydrogens yield a single signal.
In chiral compounds such as 2-butanol, replacing the methylene hydrogens at C3 produces a pair of...
In chiral compounds such as 2-butanol, replacing the methylene hydrogens at C3 produces a pair of...
3.1K
01:192D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)
1.4K
Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a heteronucleus. The HSQC experiment is similar to the heteronuclear correlation experiment (HETCOR) but is more sensitive. In the HSQC spectrum, the proton chemical shift is plotted on the horizontal F2 axis, while the 13C chemical shift is plotted on the vertical F1 axis. The corresponding proton and 13C spectra are also shown. The HSQC contour plot does...
1.4K