Oxidation‐assisted structural elucidation of compounds
containing a tertiary amine side chain using liquid
chromatography mass spectrometry
Tamar Shamai Yamin
Department of Analytical Chemistry, Israel
Institute for Biological Research (IIBR), P.O.B.
19, Ness Ziona, Israel
Avi Weissberg, Department of Analytical
Chemistry, Israel Institute for Biological
Research (IIBR), P.O.B. 19, Ness Ziona, Israel.
A novel analytical technique for the structural elucidation of compounds bearing a ter-
tiary amine side chain via “in vial” instantaneous oxidation and liquid chromatography
mass spectrometry (LC‐MS) was developed. A series of lidocaine homologs and benz-
imidazole derivatives with a major/single amine representative base peak in both their
EI‐MS and ESI‐MS/MS spectra were subjected to oxidation by a 0.1% solution of
hydrogen peroxide (including several
O exchange experiments), followed by
LC‐ESI‐MS/MS analysis. The N‐oxide counterparts promoted extensive fragmentation
with complete coverage of all parts of the molecule, enabling detailed structural
elucidation and unambiguous identification of the unoxidized analytes at low
nanogram per milliliter levels.
LC‐MS/MS, oxidation, structural elucidation, tertiary amine side chain, water analysis
Mass spectrometry is commonly applied to qualitatively and quantita-
tively profile small molecules. The increased availability of high‐reso-
lution mass spectrometry (HR‐MS) for chemical analysis has
dramatically improved the detection and identification of compounds
in the fields of metabolomics, drug discovery, forensics, and environ-
Modern mass spectrometers provide accurate
measurements of the mass‐to‐charge ratios of ions with errors as
low as sub‐ppm depending on the instruments and calibration.
addition to the mass accuracy, the ability of a mass spectrometer to
faithfully measure the isotopic distribution of an ion is also important
to determine the elemental composition of a molecule. Even a high
mass accuracy and correct isotope distribution are not sufficient to
determine the unique chemical structure of a molecule based on
the mass value alone.
Evaluation of tandem MS (MS/MS) fragmenta-
tion is useful, however not always sufficient to propose structures.
For structure elucidation, which is the main bottleneck in the identi-
fication of unknown compounds,
MS/MS spectra with diagnostic
and informative product ions representing all parts of the molecule
are needed. The most common approach to determining the
structure of unknown small organic molecules is searching the molec-
ular formula against databases, such as Chemindex, NIST library, or
even Scifinder, for possible structures. Structure‐fragmentation rela-
tions have been widely studied by Niessen,
Levsen et al,
and our group.
Various algorithms that evaluate the struc-
tures of organic compounds and predict mass spectral fragments
based on these rules have been developed (Mass Frontier, Mass
Fragmenter, Advanced Chemistry Development, EPIC).
difficulties arise in cases where the ESI‐MS/MS spectrum is uninfor-
mative or exhibits poor quality, leading to insufficient evidence to
match the ESI‐MS/MS spectra to the proposed structure. Several
compounds containing an amine side chain, particularly tertiary
amines, present information‐poor mass spectra with product ions
mostly/only representing the amine‐containing residue.
of information is attributed to the strong tendency of the amine side
chain to stabilize the positive charge and leads to unavoidable ambi-
guity in the identification process. To address this challenge, we have
recently demonstrated a method for the structural determination of
benzimidazoles containing a tertiary amine side chain using multi-
stage tandem MS, in combination with fragmentation pattern
matching to an analog utilizing LC‐MS analysis.
Received: 30 January 2018 Revised: 27 February 2018 Accepted: 2 March 2018
518 Copyright © 2018 John Wiley & Sons, Ltd. J Mass Spectrom. 2018;53:518–524.wileyonlinelibrary.com/journal/jms