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Differentiation of similar octapeptides
Figure  shows the spectra of two peptides whose mass-to-charge ratios differ by only 1 m/z. The only difference in the sequence is at the C-terminus where one peptide has threonine and the other has threonine amide. The smaller fragments are identical in the two spectra, indicating that large portions of the two peptides are very similar. The larger frag- ments contain the differentiating peptides.

Determining the molecular weight of green fluorescent protein
Green fluorescent protein (GFP) is a 27,000- Dalton protein with 238 amino acids. It emits a green light when excited by ultraviolet light.

During electrospray ionization, GFP acquires multiple charges. This allows it to be analyzed by a mass spectrometer with a relatively limited mass (mass-to-charge) range. Mass deconvolution is then used to determine the molecular weight of the protein.

The upper part of the display in Figure  shows the full scan mass spectrum of GFP. The pattern of mass spectral peaks is charac- teristic of a multiply charged analyte. Each peak represents the molecule with a different number of charges. The lower display is a deconvoluted mass spectrum generated by the data system for the singly charged analyte.

Structural Determination
Another fundamental application of LC/MS is the determination of information about molecular structure. This can be in addition to molecular weight information or instead of molecular weight information if the identity of the analyte is already known.

Structural determination of ginsenosides using MSanalysis
Ginseng root, a traditional Chinese herbal remedy, contains more than a dozen biologically active saponins called ginseno- sides. Since most ginsenosides contain multiple oligosaccharide chains at different positions in the molecule, structural elucidation of these compounds can be quite complicated.

MS analysis in an ion trap mass spectrometer permits multiple stages of precursor ion isolation and fragmentation. This stepwise fragmentation permits individual fragmentation pathways to be followed and provides a great deal of structural information.

Figure  shows the full scan mass spectrum from a direct infusion of the ginsenoside Rb1. The most prominent feature is the sodium adduct ion [M + Na]+ at m/z 1131.7. MS/MS of m/z 1131.7 yields a product ion at m/z 789.7 corresponding to cleavage of a single glyco- sidic bond (Figure 2). Subsequent isolation and fragmentation of m/z 789.7 (Figure 3) yields two products: a more abundant ion at m/z 365.1 corresponding to loss of the oligo- saccharide chain (–Glc 2 –Glc), and a less abundant ion at m/z 627.5 representing the loss of a deoxyhexose sugar.

Pharmaceutical Applications

Rapid chromatography of benzodiazepines
The information available in a mass spectrum allows some compounds to be separated even though they are chromatographically unresolved. In this example, a series of benzo- diazepines was analyzed using both UV and MS detectors. The UV trace could not be used for quantitation, but the extracted ion chromatograms from the MS could be used.

The mass spectral information provides addi- tional confirmation of identity. Chlorine has a characteristic pattern because of the relative abundance of the two most abundant isotopes. In Figure , the triazolam spectrum shows that triazolam has two chlorines and the diazepam spectrum shows that diazepam has only one.

Identification of bile acid metabolites
The MS capabilities of the ion trap mass spectrometer make it a powerful tool for the structural analysis of complex mixtures. Intelligent, data-dependent acquisition tech- niques can increase ion trap effectiveness and productivity. They permit the identification of minor metabolites at very low abundances from a single analysis. One application is the identification of metabolic products of drug candidates.

This example uses the in vitro incubation of the bile acid deoxycholic acid with rat liver microsomes to simulate metabolism of a drug candidate. Intelligent, data-dependent acquisition was used to select the two most abundant, relevant ions in each MS scan. These precursor ions were automatically fragmented and full scan product ion spectra collected.

Figure A shows the base peak chromato- gram. Figure B shows the extracted ion chromatogram of the [M-H]– ion at m/z 407 corresponding to a predicted minor metabolite (cholic acid) that eluted at 9.41 minutes. The full scan MS/MS product spectrum (Figure 28C) from the ion at m/z 407 confirms the identity.

Significant time was saved because the confirming MS/MS product ion spectra were acquired automatically in the same run as the full scan MS data.



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