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Ion Composition Elucidation (ICE)

Comparison of Time-of-Flight and Double Focusing Mass Spectrometry for Reaching Tentative Identifications of Unanticipated Compounds

Andrew H. Grange1, Floyd A. Genicola2, and G. Wayne Sovocool1

1Environmental Sciences Division, NERL, U.S. EPA, PO Box 93478, Las Vegas, NV 89193-3478
2Office of Coastal Planning & Program Coordination, N.J. Department of Environmental Protection, PO Box 418, 401 E. State Street, Trenton, NJ 08625-0418

At the 50th annual meeting of the American Society for Mass Spectrometry, only this poster and one other dealt with identifying unanticipated compounds that are not on a target list.&nbsp This poster illustrates that ICE used with a double focusing mass spectrometer remains the most powerful analytical method for determining ion compositions, despite recent advances in the capabilities of oa-TOF (and FTICR) mass spectrometers.


Monitoring of drinking water using bench-top mass spectrometers could identify target compounds in a mass spectral library. But authorities will want to know the identities and the toxicities of compounds not in the library as soon as possible.&nbsp Unanticipated compounds of unknown polarity can be rapidly separated from other components of complex mixtures using chromatographic techniques and analyzed through mass spectrometry.&nbsp Determining the molecular and fragment ion compositions in a mass spectrum constrains the number of possible isomers and can lead to compound identification based on modest literature searches.


The exact mass of an ion measured with an error limit of 5 ppm that contains C, H, N, O, P, or S atoms usually corresponds to multiple possible compositions for ions higher in mass than 150 Da.&nbsp Determination of the exact masses of the +1 and +2 mass peak profiles and their abundances relative to the monoisotopic ion provide four additional measurements for rejecting incorrect compositions.&nbsp In Figure 1a are displayed calculated profiles (Gaussian distributions) for the C23H28O2Br+ ion, the three most abundant +1 and +2 ions, and the composite +1 and +2 profiles.

 Figure 1 - For further information contact grange.andrew@epa.gov

 Figure 1.&nbsp (a) Calculated profiles for the C23H28O2Br+ ion, the three most abundant +1 and +2 ions, and the composite +1 and +2 profiles.&nbsp (b) Partial m/z 415, +1, and +2 profiles plotted from chromatographic peak areas under ion chromatograms for 7 m/z ratios across each profile.&nbsp (c) Ion chromatograms for the m/z ratios at the maxima of the partial profiles.

The theoretical exact masses and relative abundances of the +1 and +2 profiles are listed under the profiles.&nbsp In Figure 1b, these values were obtained from the top portions of the mass peak profiles, which were plotted from selected ion recording data (MPPSIRD) acquired as the two isomers evident in Figure 1c eluted into a Finnigan MAT 900S double focusing mass spectrometer (1).&nbsp The chromatographic peak areas in Figure 1c provided the maxima of the partial profiles.&nbsp Each of 31 m/z ratios was monitored for 20 msec during each 1-s SIR cycle.&nbsp Each partial profile was plotted from 7 m/z ratios and 5 m/z ratios were monitored for each of two partial profiles for calibrant ions (not shown).

A profile generation model (PGM) automatically determines the correct ion composition by rejecting all compositions with calculated values of these three exact masses and two relative abundances that are inconsistent with the measured values (2).&nbsp Use of MPPSIRD and the PGM in concert is Ion Composition Elucidation (ICE).&nbsp Table 1 lists possible compositions for a 6 ppm error limit about the measured mass of an arsenic containing compound found in a monitoring well at a landfill.

Table 1 - For further information contact grange.andrew@epa.gov

The two additional exact mass measurements and two relative abundance measurements provided compelling evidence for the last composition, the only one for which all five measured and calculated values agreed.&nbsp Also evident from the table, accurately measured relative abundances were more discriminatory against incorrect compositions than the two additional exact masses.

Instrument Requirements

To measure the three exact masses and two relative abundances listed in Table 1 for ions produced from eluting compounds, the mass analyzer must provide rapid scanning, accurate masses, a wide linear dynamic range, and resolving power sufficient to distinguish between analyte and interfering ions.

Chart - For further information contact grange.andrew@epa.gov

Two Real-World Examples

An Arsenic Containing Compound

The mass spectrum for a trace-level compound in an extract of water from a monitoring well at a landfill displayed only m/z 182 and 167 ions above the chemical noise.&nbsp The compound was hypothesized to be 2-methyl-1,3,2-dithiarsolane (structure shown in Table 1.&nbsp A conservative error limit of 6 ppm was assumed for MPPSIRD with 10,000 resolving power (10% valley).

But what if the presence of an arsenic atom was not suspected? &nbsp Consideration of C, H, N, O, F, P, and S atoms and an exact mass correct to within 5 ppm determined by oa-TOF MS would provide four compositions:&nbsp H6O3S4, HNOF3P2S, CHN2O3P3, and C3HNOFS3.&nbsp The PGM would find no viable compositions based on three exact masses and two relative abundances.&nbsp However, the relative abundances would lead to the correct composition deductively.&nbsp In Table 1, the %+2 value of 8.54% suggests two S atoms, which contribute 1.58% to the %+1 value.&nbsp The remaining %+1 of 3.14% corresponds to three C atoms.&nbsp Two S atoms and three C atoms account for 100 out of 182 Da.&nbsp One or more monoisotopic atoms are present.&nbsp As has an atomic mass of 75 Da and 7 H atoms would account for the remaining mass.&nbsp The composition C3H7S2As would then be confirmed experimentally.&nbsp

MPPSIRD provided data that would lead deductively to the correct composition, even though an element was overlooked.&nbsp A single exact mass provided by oa-TOF MS would provide no such clues.

A High-mass Disinfection Byproduct

The low resolution mass spectrum in Figure 2a with two mass peaks at m/z 415 and 417 visible above the chemical noise suggested a mono-brominated compound might be present in a chlorine-disinfected, well-water extract.  

Figure 2 - For further information contact grange.andrew@epa.gov

Figure 2.&nbsp (a) Raw mass spectrum corresponding to the maximum in the m/z 415 ion chromatogram in Figure 1c, and (b) the background subtracted mass spectrum.

Related ions with m/z 430 and 432 became apparent in the background subtracted mass spectrum in Figure 2b for which no library matches were found.&nbsp The exact mass of the apparent molecular ion was determined to be 430.15123 Da.&nbsp Assuming the presence of a single Br atom, this exact mass with a presumed error limit of 5 ppm for oa-TOF MS corresponds to 41 possible compositions.&nbsp For the fragment ion (m/z 415.12744 5 ppm), 50 compositions would be possible.&nbsp Table 3 provides the last seven possible compositions listed by the PGM for both the apparent molecular ion and the fragment ion using MPPSIRD and 20,000 resolving power (10% valley).&nbsp

 Table 3 - For further information contact grange.andrew@epa.gov

The partial profiles for the m/z 415 and its isotopic profiles are shown in Figure 1b.&nbsp For the list of possible compositions based on the exact masses of these ions, the relative abundance of the +1 profile arising primarily from 13C atoms rejected all but the correct composition in both cases.

ICE provided a unique composition for the molecular and fragment ions, while oa-TOF would have left 41 and 50 viable compositions, respectively.

Library Searches Based on One Composition

With a single composition to consider, C24H31O2Br, for the apparent molecular ion, chemical reasoning and searches of the chemical and commercial literature can lead to compound identification.&nbsp Chlorination of the well water containing bromide ions could brominate organic compounds.&nbsp The structure of Quinbolone, an anabolic steroid, is shown in Figure 2b and has three possible allylic bromination sites, which can account for the two isomers observed in the ion chromatograms in Figure 1c.&nbsp Substitution of a Br atom for an H atom would provide the observed composition.&nbsp A feed lot was located near the well and anabolic steroids are often used to stimulate growth.&nbsp Purchase of Quinbolone, its chlorination in the presence of bromide ions, and examination of the mass spectra of the products would be logical next steps in the identification process for this compound.


MPPSIRD with a double focusing mass spectrometer provides more accurate measurement of exact masses and relative abundances than the current generation of oa-TOF mass spectrometers.&nbsp Consequently, MPPSIRD is better able to determine compositions of ions in mass spectra that can lead to compound identifications.


Full-size double focusing mass spectrometers cost about $500,000 and have large foot prints.&nbsp However, a bench-top double focusing mass spectrometer with a price more similar to those of oa-TOF instruments is commercially available.&nbsp With lower mass resolution than the larger instruments, but the same linear dynamic range advantage, would this instrument also be superior to oa-TOF MS for determining ion compositions?

Multiple MPPSIRD experiments are required to determine the composition of an ion, while oa-TOF MS can acquire data for all prominent ions in one mass spectrum.&nbsp oa-TOF instruments will become more useful for determining ion compositions as their specifications for mass accuracy, linear dynamic range, and resolving power improve.&nbsp Ultimately, the two types of instruments may compliment each other.&nbsp Determination of molecular ion compositions using MPPSIRD will set limits for the elements and atoms of each element, which will in turn limit the list of possible compositions provided by an oa-TOF MS for the fragment ions.&nbsp Fewer experiments would be needed to reveal the compositions of the prominent fragment ions.&nbsp Knowledge of the fragment ion compositions limits the number of possible isomers.

ICE + Containment

Only ECB now performs ICE.&nbsp This lab is not equipped to work with unanticipated compounds that could be extremely toxic.&nbsp ECB is ready and willing to transfer ICE technology to containment labs within secure facilities.&nbsp If necessary, ECB will adapt the ICE code for the data systems of other models of double focusing mass spectrometers.&nbsp The labs expected to identify compounds added to water supplies would then have a powerful new analytical tool for doing so.


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Notice:&nbsp The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded this research and approved the abstract of this poster.&nbsp The actual presentation has not been peer reviewed by EPA.

Analytical Environmental Chemistry
ICE Home Page

Environmental Sciences | Office of Research & Development
National Exposure Research Laboratory
Author: Andrew Grange
Email: grange.andrew@epa.gov

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