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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.  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.
Introduction
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.  Unanticipated compounds of unknown polarity can be rapidly separated from other components of complex mixtures using chromatographic techniques and analyzed through mass spectrometry.  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.
Methods
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.  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.  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.  (a) Calculated profiles for the C23H28O2Br+ ion, the three most abundant +1 and +2 ions, and the composite +1 and +2 profiles.  (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.  (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.  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).  The chromatographic peak areas in Figure 1c provided the maxima of the partial profiles.  Each of 31 m/z ratios was monitored for 20 msec during each 1-s SIR cycle.  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).  Use of MPPSIRD and the PGM in concert is Ion Composition Elucidation (ICE).  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.
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.  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.
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.  The compound was hypothesized to be 2-methyl-1,3,2-dithiarsolane (structure shown in Table 1.  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?   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:  H6O3S4, HNOF3P2S, CHN2O3P3, and C3HNOFS3.  The PGM would find no viable compositions based on three exact masses and two relative abundances.  However, the relative abundances would lead to the correct composition deductively.  In Table 1, the %+2 value of 8.54% suggests two S atoms, which contribute 1.58% to the %+1 value.  The remaining %+1 of 3.14% corresponds to three C atoms.  Two S atoms and three C atoms account for 100 out of 182 Da.  One or more monoisotopic atoms are present.  As has an atomic mass of 75 Da and 7 H atoms would account for the remaining mass.  The composition C3H7S2As would then be confirmed experimentally. 
MPPSIRD provided data that would lead deductively to the correct composition, even though an element was overlooked.  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.  (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.  The exact mass of the apparent molecular ion was determined to be 430.15123 Da.  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.  For the fragment ion (m/z 415.12744 ñ 5 ppm), 50 compositions would be possible.  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). 
The partial profiles for the m/z 415 and its isotopic profiles are shown in Figure 1b.  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.  Chlorination of the well water containing bromide ions could brominate organic compounds.  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.  Substitution of a Br atom for an H atom would provide the observed composition.  A feed lot was located near the well and anabolic steroids are often used to stimulate growth.  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.
Conclusion
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.  Consequently, MPPSIRD is better able to determine compositions of ions in mass spectra that can lead to compound identifications.
Speculations
Full-size double focusing mass spectrometers cost about $500,000 and have large foot prints.  However, a bench-top double focusing mass spectrometer with a price more similar to those of oa-TOF instruments is commercially available.  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.  oa-TOF instruments will become more useful for determining ion compositions as their specifications for mass accuracy, linear dynamic range, and resolving power improve.  Ultimately, the two types of instruments may compliment each other.  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.  Fewer experiments would be needed to reveal the compositions of the prominent fragment ions.  Knowledge of the fragment ion compositions limits the number of possible isomers.
ICE + Containment
Only ECB now performs ICE.  This lab is not equipped to work with unanticipated compounds that could be extremely toxic.  ECB is ready and willing to transfer ICE technology to containment labs within secure facilities.  If necessary, ECB will adapt the ICE code for the data systems of other models of double focusing mass spectrometers.  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:  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.  The actual presentation has not been peer reviewed by EPA.