Accurate mass measurements have been used for the formula identification of unknown compounds since the advent of high-resolution mass spectrometers. This simple but elegant method of formula identification relies on the fact that each unique formula has a unique accurate mass (4–6). Depending upon the inherent mass accuracy for a given instrument, all formulas falling within the instrument's mass error range need to be considered as viable candidates. For example, the formula search on an instrument capable of obtaining a mass accuracy of 1 ppm results in a list of 34 formula candidates for an unknown compound at 500 Da containing the elements C, H, N, O, S, and Cl. The formula candidates can be pared down by imposing chemical constraints, such as limiting the possible elements in the formula search, the minimum and maximum number of atoms for each element, the electron state, and any other complementary knowledge of the unknown sample. Indeed, it is well known that mass accuracy alone generally is not enough to obtain a unique formula (3), even on the highest resolution instruments.

Isotope ratio measurements also have long been used to assist in reducing the number of formula candidates for accurate mass measurements (7). Like accurate mass, every unique formula has a unique isotope distribution. For an isotopic abundance distribution accuracy (spectral accuracy) of 98%, 95% of the formula candidates can be eliminated at 3 ppm mass accuracy, providing the performance of a hypothetical system capable of 0.1 ppm mass accuracy (6). If the isotopic abundance distribution error, or spectral accuracy, could be improved to better then 99%, this could improve substantially the ability to uniquely identify the formula even on systems with very modest mass accuracy. In general, such high spectral accuracy is difficult to obtain, primarily because of the instrument-specific distortions in the measured instrument line-shape, which make accurate isotope pattern matching inherently inaccurate.

Recent work employing a novel approach to mass spectral calibration also provides calibration of the instrument line-shape to a known analytical function, dramatically improving both the mass accuracy and the spectral accuracy (2), even for unit resolution quadrupole systems. The method requires that one or more calibration ions that are relatively close in mass (for example, 50 Da) be measured within a short time period (a couple of hours to days, depending upon the specific instrumentation) for best results. The same approach applied to high-resolution mass spectrometry (MS) shows only an incremental improvement in mass accuracy (8), which is probably limited by the fundamental performance limits of the instruments and the calibration procedure used. Any further improvements in formula identification for a given mass accuracy might be gained from methods that improve the instrument line-shape calibration. Improvement of the instrument line-shape calibration has been shown to make formula identification on unit resolution systems possible (3) and should further benefit high-resolution systems as well.

Instrument Calibration

While it has been shown that including line-shape calibration for high-resolution systems can improve formula identification (8), frequent recalibration with known standards is still required for optimum performance. This is due to the short- and long-term stability inherent in all mass spectrometers. A number of different calibration approaches are used, all of which have advantages and disadvantages. One approach involves using dual spray ion sources that continually introduce a calibration standard to allow for constant recalibration. One drawback of dual spray, beyond the increased cost and complexity of the hardware, is that either the measurement duty cycle time of the instrument is reduced if the calibration standard is introduced periodically, or the unknown mass spectrum might be contaminated if it is introduced simultaneously. Another drawback is that the calibrant is not always optimum for the ion of interest. For best performance, the calibrant or "lock mass" should be as close in mass as possible to the analyte ion. This is not always convenient or even possible when many analytes of varying masses are to be measured.