In full scan, changes in mass to a molecular ion are used to determine changes to the parent compound (see Table 6.1). This information, plus knowing the source of the metabolite, allows for determination of overall changes to the compound.
• Isotope patterns are characteristic of compounds and can be used to identify metabolites. For example, for a parent molecule that includes a chlorine atom, an isotope pattern of M, M + 2 with a ratio 3:1 is expected. So, if the chlorine atom is retained in the drug/metabolite, the same pattern is expected (see Table 8.6 for other elements).
• High resolution MS can be used to identify the elements present in the molecule and, in many cases, assess the exact nature of the modification. For example, an addition of 14 Da can be due to methylation (+CH2) or oxidation and desaturation (+O-2H). In high resolution MS, one is looking at the difference between addition of 14.0266 Da (12.0107 + (2 x 1.0079) for +CH2) and 13.9841 Da (15.9994 - (2 x 1.0079) for O-2H).
• In-source fragmentation forms fragment ions that could lead to the wrong interpretation of the molecular ion. For example, insource fragmentation of glucuronide metabolites leads to cleavage of glucuronic acid (-176 Da) in the source, resulting in an increased abundance of molecular ions of the aglycan metabolite.
• Formation of adducts leads to formation of molecular ions that correspond not to [M + H]+, but to [M + NH4]+ (addition of 17.0306 amu), [M + Na]+ (addition of 21.9818 amu), and [M + K]+ (addition of 38.0904 amu).
• Knowing the expected metabolites from a source helps with predicting the possible modifications. For example, glucuronide metabolites are not expected when using liver microsomes without the necessary cofactors (i.e., uridine diphosphate glucuronic acid).
Double bond equivalent (DBE; known also as degree of unsaturation) is the number of double bonds or rings in a molecule. It is derived in the following way:
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