Study of Free Radical Fragment Ions Generated from ESI-CID-MS-MS Using LTQ and LTQ Orbitrap Mass Spectrometers


The Application Notebook

The Application NotebookThe Application Notebook-09-01-2008
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Studies of odd-electron CID behaviors reveal that free radical fragmentation is structure-dependent and is directly correlated with the functional groups that stabilize the newly-formed free radicals.

Guifen Xu1 , Tom Huang1 , Jennifer Zhang2 , Thomas D. McClure2 , and Shichang Miao1* ,1 PKDM Department, Amgen SSF; * Analytical Chemistry and DMPK, ChemoCentryx Inc.,2 Thermo Fisher Scientific

Studies of odd-electron CID behaviors reveal that free radical fragmentation is structure-dependent and is directly correlated with the functional groups that stabilize the newly-formed free radicals.

Mass spectrometry has become an indispensable tool for structural analysis in drug discovery and development. A key element in analysis of structures by mass spectrometry is to identify correct fragmentation pathways. Collision induced dissociation (CID) has been extensively used for structure elucidation to determine fragmentation pathways.

Figure 1

CID in the ESI and APCI modes has been found to generate mostly even-electron fragments, while it has been occasionally reported to form odd-electron free radical ions. We studied a series of aromatic and non-aromatic compounds, such as sulfonamides, amides, aromatic t-Bu compounds, aromatic ether, oxime ethers, and pyrimidines using the LTQ and LTQ Orbitrap mass spectrometers (Thermo Scientific, San Jose, California) to determine the structural requirement and the fragmentation mechanisms for free radical CIDs.

Experimental Methods

Chemicals and Materials

All test compounds were purchased from Sigma-Aldrich (Milwaukee, Wisconsin) or Maybridge (Morris Plains, New Jersey) and were prepared in 40 μM with 1:1 methanol-H2O before ESI-CID-MS-MS analysis without further purification.

LC–MS-MS Conditions

All samples were first analyzed using ESI on the LTQ coupled with pumps and autosampler under standard conditions: capillary temperature, 325°C; source voltage, 6000 V. Helium was used as the collision gas. The MS-MS parameters were: isolation width, 2; and collision energy, 35%. Compounds with good MS and MS2 signals were further analyzed using ESI-LTQ Orbitrap coupled with the Thermo Scientific Accela U-HPLC system for accurate mass measurement. The MS-MS parameters were the same as those used for the LTQ.


A series of aromatic and non-aromatic compounds such as sulfonamides, amides, phenols, oximes, and pyrimidines were analyzed for their CID fragments, and the molecular compositions of the key fragments were identified through accurate mass measurement using the LTQ Orbitrap mass spectrometer.

Shown in Figure 1 is a representative MS2 fragment ion mass spectrum of one of the compounds.


For sulfonamides, in general, when the sulfonamide nitrogen was attached to an aromatic ring, free radical fragment ions involving the aromatic amine due to the S-N bond cleavage were observed, unless more facile CID pathways were present and prevailed in the structure. For non-aromatic sulfonamides, free radical fragments were not observed in the test compounds.

Aromatic t-Bu and ether compounds normally generated a free radical fragment ion resulting from the loss of the methyl or ethyl free radical. Free radical fragments were observed more rarely in the selected amides, pyrimidine, and oxime compounds.

The assignment of these free radical ions was all supported by the accurate mass measurement on the LTQ Orbitrap with a mass accuracy less than 5 ppm. One common theme among these compounds that showed free radical fragment ions is that the newly formed free radical is located at a position next to an aromatic ring which stabilizes this free radical.


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