One application of this calibration technique is shown in Figure 4 for liquid chromatography- mass spectrometry (LC/MS). In
this example a typical unit mass resolution triple quad instrument is used to screen for the metabolites of the drug verapamil
after microsome incubation. Since the calibration was performed after the data acquisition, the known compound (verapamil)
could be used as the calibration ion for the entire run. The chromatographic peak eluting before verapamil is suspected of
being its demethylation metabolite. To confirm this, the accurate mass of the calibrated monoisotope peak at 441.2742 Da compares
favorably to the theoretical mass of 441.2753 Da. This corresponds to an error of about 1 mDa or 2.5 ppm. More typically,
we have found that we can routinely measure the mass accuracy on such instruments in the 5-10 ppm range.
Figure 5 shows an example of the calibration applied to an environmental gas chromatography – mass spectrometry (GC/MS) application
for identifying pesticides using an Agilent MSD detector (single quad mass spectrometer). A commercial calibration mixture
was used to externally calibrate the MS. In the blind test, the Monsanto pesticide PCB 209 (CAS number 2051-24-3) was calculated
as having a mass of 493.6855 Da as compared to the theoretical mass of 493.6885 Da. This corresponds to an error of 3 mDa
or about 6 ppm, a mass accuracy observed for other pesticides in the mixture as well.
Another application area includes the analysis of a known compound in spectra containing a complex background. For example,
in metabolite identification, drug metabolites need to be identified in extremely complex bile matrices. Conventional extracted
ion chromatograms (XIC) are not selective enough, and background interferences create many false positives. Each positive
may need to be further investigated with techniques such as MS/MS or C14-labeled compounds.
Due to the comprehensive calibration of the MSIntegrity systemn, we can instead calculate an XIC by using a narrower and more
accurate mass window, and the entire isotope profile can be used as a highly selective mass filter. The accurate mass profile
XIC (AMPXIC) provides a dramatic improvement by rejecting interferences including many that actually fall within the same
mass window. An example is shown in Figure 6 with verapamil incubation. This same approach can be applied to impurity, degradant,
and compound identification studies.
The ability to obtain high mass accuracy on unit mass resolution instruments combined with peak shape calibration has the
potential to enhance or even create entirely new methods of analysis. One benefit of this approach is that it allows the input
of calibrating-data from various instruments (including instruments of various types made by different manufacturers) to output
the same results in terms of peak shapes and positions regardless of instrument tune and other variables (mass spectral alignment).
Thus, applications that are heavily dependent on identifying subtle differences between complex samples, such as biomarker
discovery and proteomics, can be dramatically improved.
The MSIntegrity approach to MS calibration can dramatically extend the utility of both new and existing unit mass resolution
systems by allowing high mass accuracy measurements to be performed for compound identification or verification. In addition,
the calibration improves the form and consistency of the data from any mass spectrometer.