Delineation of Isomers by the ¹³C Shifts in Ion Mobility Spectra

Abstract

Mass spectrometry (MS) and isotopes were intertwined for a century, with stable isotopes central to many MS identification and quantification protocols. In contrast, the analytical separations including ion mobility spectrometry (IMS) largely ignored isotopes, partly because of insufficient resolution. We recently delineated various halogenated aniline isomers by structurally specific splitting in FAIMS spectra. While this capability hinges on the 13 C shifts, all preceding studies leveraged 37 Cl or 81 Br to enhance the differentiation. However, such abundant heavy isotopes are absent from typical organic compounds. With single I isotope, iodinated organics generate similar isotopic envelopes dominated by the 13 C atoms. Here, we distinguish the three monoiodoaniline isomers based on the shifts solely for one or two 13 C atoms. The differentiation may be somewhat improved using multipoint peak position descriptions for more reproducible shifts. The interisomer order of shifts differs from those for chlorinated or brominated analogues, showcasing the specificity of approach. We also investigated the mass scaling of isotopic shifts, encountering divergent trends for different structural families.

Isomer and Conformer-Specific Mass Distribution-Based Isotopic Shifts in High-Resolution Cyclic Ion Mobility Separations

“…While there have been fewer demonstrations of isotopologue and isotopomer separations with TWIMS, it is important to discuss contributions from groups utilizing other IMS-based techniques, such as field asymmetric ion mobility spectrometry. Additionally, it is necessary to mention that atmospheric pressure DTIMS has also been utilized to separate isotopologues of small molecules (e.g., acetone and benzene-like compounds). − Notably, chloride isotopes, glycine isotopomers/isotopologues, and di-alanine isotopomers were resolved in early demonstrations of isotopic separations in FAIMS. ,, More recently, FAIMS-based studies of halogenated aniline rings (e.g., mono/di/tri-substituted aniline rings containing chlorine, bromine, and iodine atoms) have revealed that elemental and structurally specific isotopic shifts, as well as these isotopic shifts, are additive in nature. ,− While the physical nature of the FAIMS-based isotopic shifts remains unknown, the authors describe them as likely being a result of center of mass shifts (i.e., mass distribution-based shifts). ,− However, the unknown extent of ion heating at the high electric fields in FAIMS convolutes analysis, potentially contributing to the observed separation. Clearly, more future work is necessary to compare isotopic shifts in FAIMS with those in atmospheric DTIMS and TWIMS-based separations.…”

High-Definition Ion Mobility/Mass Spectrometry with Structural Isotopic Shifts for Nominally Isobaric Isotopologues

“…This paradigm was initially developed utilizing the He/CO 2 buffers and protonated haloanilines with up to three Cl, Br, or I atoms at varied ring positions, producing three or six isomers ( m ∼ 130–330 Da). − These species display MS features above the base peak by +1 Da (dominated by 13 C) and +2 Da (mostly due to 37 Cl, 81 Br, or 13 C 2 for the iodoanilines with monoisotopic halogen). Some isomers were distinguished by single E C shifts, and all were delineated by 2-D ( 13 C/ 37 Cl or 13 C/ 81 Br) shifts. , The shifts for all isotopic groups (save 13 C 2 in few cases) − were additive as in normalΔ E normalC ( C 13 C 37 l ) = normalΔ E normalC ( C 13 ) + normalΔ E normalC ( C 37 l ) This permitted averaging several Δ E C values in same spectrum, up to n = 4 for 13 C in tribromoanilines (between m / z of 328 and 329 for sole 13 C, 330 and 331 for 13 C on top of 81 Br, 332 to 333 for 13 C on top of 81 Br 2 , and 334 to 335 for 13 C on top of 81 Br 3 ) . Such multiplexing reduces random Δ E C errors by ideally the factor of n 1/2 , enhancing the isomer demarcation. − …”

“…Some isomers were distinguished by single E C shifts, and all were delineated by 2-D ( 13 C/ 37 Cl or 13 C/ 81 Br) shifts. , The shifts for all isotopic groups (save 13 C 2 in few cases) − were additive as in normalΔ E normalC ( C 13 C 37 l ) = normalΔ E normalC ( C 13 ) + normalΔ E normalC ( C 37 l ) This permitted averaging several Δ E C values in same spectrum, up to n = 4 for 13 C in tribromoanilines (between m / z of 328 and 329 for sole 13 C, 330 and 331 for 13 C on top of 81 Br, 332 to 333 for 13 C on top of 81 Br 2 , and 334 to 335 for 13 C on top of 81 Br 3 ) . Such multiplexing reduces random Δ E C errors by ideally the factor of n 1/2 , enhancing the isomer demarcation. − …”

“…With |Δ m | ≤ 9 mDa, these isotopologues fully merge in ion trap MS even in the ultrazoom mode ( R MS ∼ 1000). − Besides the loss of information about their E C shifts, such contamination affects the Δ E C values ascribed to the major isotopologues at unit masses. The deviation must be minor for haloanilines with single isotopologues contributing >90% of signal at each unit mass, − but not species with comparably abundant nominal isobars. For example, the peak at m / z = 167 for deprotonated phthalic acids (C 8 H 5 O 4 – , base m / z = 165) comprises the isotopologues with 18 O and 13 C 2 in a 70:29 ratio.…”

“…We preferred the He/N 2 buffers for conventional FAIMS separations and He/CO 2 for isotopic analyses. − Such buffers containing light and heavy gases tend to augment the resolution because of non-Blanc effects (deviations from the Blanc’s law of diffusion at high electric fields, prominent in mixtures of disparate gases), but are incompatible with ultrahigh vacuum pumping of Orbitrap MS. Hence, combining high-definition FAIMS with Orbitrap MS necessitated the pumping modifications and/or a tandem electrodynamic ion funnel interface (IFI) to dilute He with heavier gases .…”