Elemental composition from accurate m/z determinations

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  • Published: Jul 1, 2014
  • Channels: Atomic / Base Peak
thumbnail image: Elemental composition from accurate <I>m</I>/<I>z</I> determinations

Professor Tony Mallet of the University of Greenwich presents an essay on elemental compositions from 'accurate mass' determinations.


The table below gives the precise atomic masses of some stable isotopes which might be commonly found in organic molecules.
















A consequence of this is that two molecules of identical nominal (integral) mass and different elemental composition, such as HCOOH and CH3OCH3 will differ significantly; e.g. 46.0054 and 46.0340.

Any mass analyser which can operate with a resolving power greater than 1600 will be able to distinguish between these two species.

{Resolving Power required = 46/(46.0340-46.0054)= 1608}

It follows that, if a sufficiently precise and accurate measurement of the m/z of an ion can be obtained, the elemental composition(s) corresponding to this value can be deduced.

Obtaining a Precise and Accurate Measurement for the m/z of an ion.

The shape of the envelope for the signal of an ion depends on the nature of the analyser. Magnetic instruments produce Gaussian curves while ion traps and tof analysers tend to give peaks with increased width at the base.  The value of the m/z is given by calculating the centroid of the peak. For a precise measurement it is essential that a symmetrical peak is produced; if there is any contribution from another component of identical integral m/z or if the peak shape is distorted due to poor tuning, an imprecise centroid will be obtained.


Note how the centroid will be shifted in B compared to the symmetrical peak A.

The absolute resolving power of the analyser is not the determining factor but sufficient must be used to obtain a symmetrical peak. Remember that for most analysers increasing the resolving power leads to a rapid loss of sensitivity and a proper balance needs to be maintained. The normal practice is for the data system to digitise the signal and from this data to reconstruct a peak. The rate of digitisation needs to be matched to the scan rate of the instrument so that sufficient points are measured over the full width of the peak (from 12 to 20 are recommended).

The next factor, after precision, is the accuracy of the determination. For this not only must the ?normal calibration of the m/z scale be performed but reference standards should be present which give two or more ions with known m/z values which bracket the unknown. Some refer to these references as ?lock masses . The nominal mass scale is then adjusted by application of the two known accurate mass components. This use of these internal standards, can be avoided by the use of external standards if the stability of the instrument is sufficient.  In practice it is the choice of reference which can lead to the greatest difficulty. It depends on how the sample is being introduced; is a probe being used with EI/CI or FAB, or is a chromatographic separation being used on-line?  The optimum results are obtained if a roughly equal intensity is detected from the unknown and the reference peaks. Obviously this becomes more difficult when a varying concentration of analyte is present, as in a chromatographic inlet system.




PFK, Heptacosa.


PEG, PPG etc


Compounds of similar structure

GC with EI

Column Bleed

It is helpful to take a number of determinations of the m/z value as this will permit an estimate of the error limits of the overall process.

Accurate m/z measurements are made on a variety of instruments but the most commonly used are double focussing magnetic and time-of-flight analysers, the latter combined with an orthogonal quadrupole analyser. The most precise results at very high resolving powers come from experiments on Fourier Transform Ion Cyclotron Resonance Spectrometers. The least used are Quistor Traps and Quadrupoles.


As the m/z value increases the number of possible elemental compositions which will fit a measurement with a known error range, also increases. Two scales of error are in common use, one is the ppm (part-per-million) scale and the other mmu (milli-mass units). Modern instruments can make measurements to within 5 ppm routinely and some can achieve better than 1 ppm.

{At mass 500 2 ppm is equivalent to 1 mmu.}

The graph below shows how the number of possible ?fits increase with an m/z of 600 as the error of determination increases. It is clear that above m/z 600 the use of this technique to prove a structure becomes increasingly invalid; the only proof will be that a suggested structure is wrong!

In order to limit the number of possible elemental compositions it is helpful to apply a number of commonsense rules. The data system can be told to ignore all impossible formulae such as those with insufficient hydrogen atoms. The list of elements to include and their maximum number can also be defined. Use all other available information such as that rising from the observed isotope ratios of the molecular ion. Remember that with ESI, not only can there be a protonated ion but also ions cationised with Na or K, are often seen.

The table above shows a typical print-out from a data system. The elements (C, H, O and S) have been defined with maximum numbers and the ion found at m/z 554.4532 has been analysed. Three possible ?fits , within an error of 10 ppm, are shown with the closest being the one with a single 32S present. This should show in the isotope pattern at the molecular ion. The table also shows the double-bond-equivalent (DBE) which will help eliminate improbable compositions.


  • Tune the instrument to produce well shaped peaks.
  • Choose a resolving power which separates any interferences from the chosen peak.
  • Scan at a rate which ensures that the data system will be able to define a peak properly.
  • Introduce appropriate internal reference materials.
  • Eliminate as many unlikely fits as possible.
  • Use all other information regarding possible elemental composition (e.g isotopic patterns).


  • Bieman K. Utility of Exact Mass Measurement. Methods in Enzymology 1990, 193, 295-305.
  • Tyler AN, Clayton E, Green BN.  Exact Measurement of Polar Organic Molecules at Low Resolution using Electrospray Ionisation and a Quadrupole Mass Spectrometer. Anal. Chem. 1996, 68, 3561-3569.
  • Eckers C, Wolff JC, Haskins NJ, Sage AB, Giles K, Bateman R. Accurate Mass Liquid Chromatography Mass Spectrometry on Orthogonal Acceleration Time-of-Flight Mass Analysers using Switching between Separate Sample and Reference Sprays. 1 Proof Concept. Anal. Chem. 2000, 72, 3683-3688.
  • Wolff JC Eckers C, Sage AB, Giles K, Bateman R. Accurate Mass Liquid Chromatography Mass Spectrometry on  Quadrupole Orthogonal Acceleration Time-of-Flight Mass Analysers using Switching between Separate Sample and Reference Sprays. 2. Applications. Anal. Chem. 2001, 73, 2605-2612.
  • Methodology for accurate mass measurement of small molecules. Best Practice Guide. LGC Ltd.,   London. 2003
  • Bristow AWT and Webb KS. Intercomparison Study on Accurate Mass Measurement of Small Molecules in Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2003, 14(10), 1086-1098.
  • Wolff JC, Thomson LA, Eckers C. Identification of the 'wrong' active pharmaceutical ingredient in a counterfeit Halfan drug product using accurate mass electrospray ionisation mass spectrometry, accurate mass tandem mass spectrometry and liquid chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 215-221.
  • Wolff J-C, Fuentes TR, Taylor J. Investigations into the accuracy and precision obtainable on accurate mass measurements on a quadrupole orthogonal acceleration time-of-flight mass spectrometer using liquid chromatography as sample introduction. Rapid Commun. Mass Spectrom. 2003, 17, 1216-1219.
  • Thompson CM, Richards DS, Fancy S-A, Perkins GL, Pullen F, Thom C.  A comparison of accurate mass techniques for the structural elucidation of fluconazole.  Rapid Commun. Mass Spectrom. 2003, 17, 2804-2808{The last three references illustrate different approaches to the measurement of accurate mass.}

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