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Ion Composition Elucidation (ICE)  


Andrew H. Grange and G. Wayne Sovocool

U.S. EPA, National Exposure Research Laboratory, PO Box 93478, Las Vegas, NV 89193-3478

Especially Noteworthy

ICE tentatively identified a compound for which 10 library matches were plausible for compounds producing three different molecular ions.


Identifying compounds found in the environment without knowledge of their origin is a very difficult analytical problem.  Comparison of the low resolution mass spectrum of a compound with those in the NIST or Wiley mass spectral libraries can provide a tentative identification when the mass spectrum is free of interferences, at least several prominent ions are observed in the mass spectrum, the mass spectrum is in the library, and only one plausible match is found.  Because these libraries contain only 390,000 mass spectra (1) compared to the 16 million compounds that have been synthesized or isolated from natural sources (2), most compounds are not found in the libraries.  In addition, most compounds are ionic, too polar, too thermolabile, or too high in mass to traverse a GC column or to volatilize from a probe.  For these compounds, liquid sample introduction with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) provides few fragment ions for pattern matching, and adduct ions complicate the mass spectra.  Commercial ESI and APCI mass spectral libraries are not available.  Consequently, low resolution mass spectrometry cannot identify most compounds.

Conventional high resolution mass spectrometry provides exact masses for ions formed from compounds volatilized from a probe or introduced by infusion.  A unique ion composition is usually determined for ions containing C, H, N, O, P, or S atoms with masses less than 150 Da and an error limit of 6 ppm for the mass determination.  However, most compounds provide ions with masses higher than 150 Da.  Ion Composition Elucidation increases the mass limit for determining ion compositions to 600 Da by acquiring data in a different manner that provides smaller error limits, and by considering the isotopic masses and abundances of 13C, 2H, 15N, 17O, 18O, 33S, and 34S atoms.

Mass Peak Profiling from Selected Ion Recording Data (MPPSIRD)

Selected ion recording provides a series of ion chromatograms for m/z ratios across part or all of the mass peak profiles for two calibration ions and up to four analyte ions (3).  The polarity of a voltage in the ion source is reversed for 5 s before and after the chromatographic peak elutes to induce baselines in the ever-present calibrant ion chromatograms, thereby creating a simulated chromatographic peak (left-most ion chromatogram in Figure 1a). 

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

Figure 1.

The areas between the baseline excursions are integrated and plotted to provide partial or full profiles for both calibrant and analyte ions (Figure 1b).

MPPSIRD provides several important advantages over full scanning.

Profile Generation Model (PGM)

A Profile Generation Model (PGM) is used to plan experiments and interpret the data (4).  The PGM lists the ion compositions possible for an exact mass and its error limits.  When multiple compositions are possible, the exact masses and abundances of the mass peak profiles heavier by 1 and 2 Da that arise from +1 and +2 ions containing heavier isotopes are also considered.  By comparing up to five pairs of measured and calculated values for each possible composition (three exact masses and two relative abundances), rather than a single exact mass, the PGM greatly reduces the number of possible compositions for an ion.

Three Applications of ICE

Rejecting Multiple Library Matches

In Figure 2a is a background-subtracted mass spectrum for a compound in an extract of 12 L of a tertiary sewage treatment plant effluent.

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

Figure 2.

The extract contained dozens of compounds at ultra-trace levels and coelution of compounds often resulted in mass spectra with extraneous or missing mass peaks.  The other six mass spectra (Figure 2b-g) are NIST matches.  The isomers in parentheses also provided plausible matches.  Three ion compositions for the apparent molecular ion (m/z 133) are represented (C7H7N3, C8H7NO, and C9H11N).  Determining the correct composition of this ion rejected most matches.

Figures 3a - 3c illustrate the stepwise procedure to determine the correct composition.

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

Figure 3.

For Figure 3a, a wide mass range (2000 ppm) was surveyed using a mass increment of 100 ppm between the m/z ratios and a mass resolution of 3000.  Three points delineated each profile, and two profiles were observed.  The ion chromatogram insets for the maximum on each profile indicated that the lower-mass profile was due to an ever-present ion (probably 13C2CF5+ from the calibrant, perfluouokerosene) and the higher-mass profile was due to the analyte ion, since a chromatographic peak was observed.  The exact mass was estimated as the weighted average of the three points on the profile and used as the center mass in the SIR descriptor used to acquire the data plotted in Figure 3b.  Ten points delineated the profile acquired with 10,000 resolution and 10-ppm mass increments.  The weighted average of eight points provided an exact mass accurate to within 6 ppm, which was entered into the PGM to provide the two possible compositions in Table I.

Table I.  Possible Compositions for an Exact Mass of 133.06403

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

To prove that the second composition was correct, full m/z 133 and +1 profiles were obtained from Figure 3c. The two measured exact masses and single relative abundance were entered into the PGM to provide Table II.

Table II.  PGM Output for Measured Values from Figure 3c

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

Either “X” next to the calculated mass defect (the non-integer part of the exact mass) or relative abundance of the first composition rejects the composition.  Only the second composition passed the three comparisons.  Only the NIST library match in Figure 2b has the second composition for the m/z 133 ion.  Of the two isomers, only 5-methyl-1H-benzotriazole is associated with a commercial use and might enter the sewage stream. It is a derivative of 1H-benzotriazole used as a corrosion inhibitor in airplane deicers.(5).  The correct composition for the m/z 133 ion reduced the number of possible compounds based on the library matches from 10 to two, and consideration of commercial uses indicated this compound should be purchased and its retention time and mass spectrum compared to the analyte to confirm its identity.

Identification of Environmental Pollutants 

Several isomeric compounds having similar mass spectra were found in an extract of municipal well water in an area where increased childhood cancer had been observed (Figure 4a).

Figure 4a - For further information contact grange.andrew@epa.gov

Figure 4a.

Figure 4b - For further information contact grange.andrew@epa.gov

Figure 4b.

The identities of the isomers were limited to those containing a tetralin core with attached cyano and alkylcyano groups by determining the ion compositions of the molecular ion and 10 fragment ions.  A search of the chemical literature revealed 1:2 styrene:acrylonitrile adducts formed in a polymerization process used in the 1970's as the likely compounds.  A standard from the process used today confirmed that three of the five isomers in the extract had the same retention times and compellingly similar mass spectra to the three isomers in the standard (Figure 4b)(6).

Determining ion compositions without mass calibrants

When analytes are introduced by infusion, ever-present analyte ions can be used as the lock-on mass.  In Figure 5a are full profiles for the protonated molecular ion (M+H)+ and +1 profile for 2-hydroxy-4-methylquinoline obtained with 10,000 resolution and in Figure 5b are the (M+H)+ and +2 profiles.

Figure 5 - For further information contact grange.andrew@epa.gov

Figure 5.

Data was acquired for 100 s as 4 śL/min of 10 ng/śL of the analyte in 1:1 methanol:water with 1% acetic acid was infused into an electrospray source.  Using full profiles limited errors to instrumental precision and the natural variation of isotope abundances for each composition.  Three standard deviations for %+1 and %+2 were estimated from 13 triplicate determinations as 3.9% of %+1 and 5.4% of %+2, respectively.  Using these error limits in the PGM to compare only relative abundances provided Table III.

Table III.  Relative Abundances for Protonated 2-hydroxy-4-methylquinoline

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

Only the correct composition for the protonated molecular ion was possible. Two additional observations are useful for determining compositions of larger ions. Exact mass differences between profiles provide compositions of composite neutral losses from the molecular ion and observation of +1 profiles at high mass resolution reveal the presence of N atoms when the ion containing a 15N atom is partially resolved from the ion containing a 13C atom (7).

Additional Applications

Analytical problems that have been solved by ICE include:

Numerous posters from ACS, ASMS, and other meetings and journal articles describing ICE and its applications are accessible at http://www.epa.gov/nerlesd1/chemistry/ice.default.


The Environmental Chemistry Branch of the EPA-Las Vegas will consider assisting in the characterization of “mystery” compounds in extracts others have prepared from environmentally important samples.  In addition, the computer code and manuals describing ICE are available for Finnigan MAT 900 or MAT 95 double focusing mass spectrometers.


Ion composition elucidation coupled two simple ideas, monitoring of multiple m/z ratios across each of several mass peak profiles and consideration of the profiles heavier in mass by 1 and 2 Da due to the natural abundances of +1 and +2 isotopes, to extend 4-fold the mass range for which unique ion compositions can be determined.  This was accomplished without hardware modifications to a double focusing mass spectrometer.  Computer code alone prepares experiments, obtains chromatographic peak areas from ion chromatograms, and plots the mass peak profiles.  The scientific basis of ICE is simple:  comparison of measured exact masses and relative abundances with calculated values based on addition of exact masses of atoms, addition of isotopic abundances, and consideration of elemental valences.  The simplicity of these ideas would make them easier to explain to a judge or jury in legal proceedings than the theory underlying energy-based spectroscopies.  Often, trace-level pollutants can be identified using ICE by greatly limiting the number of possible compounds followed by searching of the chemical and commercial literature.  The retention time and mass spectrum of one or a small number of standards can then confirm tentative identifications.  In these cases, preparative scale chromatography to obtain enough analyte for FTIR and NMR studies to identify compounds is unnecessary.


  1. http://www.wiley.com/WileyCDA/WileyTitle/productCd-0471440973.htmlExiting EPA Disclaimer

  2. http://www.cas.org/casdb.htmlExiting EPA Disclaimer

  3. Grange, A.H.; Donnelly, J.R.; Sovocool, G.W.; Brumley, W.C. Anal. Chem. 1996, 68, 553-560.

  4. Grange, A.H.; Brumley, W.C. J. Am. Soc. Mass Spectrom. 1997, 8, 170-182.

  5. http://www.engg.ksu.edu/HSRC/ag/2002/proceed/k04.pdfExiting EPA Disclaimer[PDF, 12 pp., size not available, About PDF]

  6. Grange, A.H.; Sovocool, G.W. Rapid Commun. Mass Spectrom. 1998, 12, 1161-1169.

  7. Grange, A.H.; Sovocool, G.W. Rapid Commun. Mass Spectrom. 1999, 13, 673-686.

  8. Grange, A.H.; Brumley, W.C. LC•GC 1996, 14, 978-986.

  9. Grange, A.H.; Sovocool, G.W. J. AOAC Internat. 1999, 82, 1443-1457.

  10. Snyder, S.A.; Kelly, K.L.; Grange, A.H.; Sovocool, G.W.; Snyder, E.M.; Giesy, J.P. in Pharmaceuticals and Personal Care Products in the Environment: Scientific and Regulatory Issues; Daughton, C.G. & Jones-Lepp, T., Eds.; Symposium Series 791; Amer. Chem. Soc.: Washington, DC, Ch. 7, 116-141.

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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