MSMS Data Product Ion Scans

Product ion scans involve selecting the ion of interest, the molecular ion in most cases, in the first quadrupole and following a fragmentation in the ion source. The fragment ions are then detected in the mass analyzer. See Table 6.2 for some common neutral loss masses for product ion scans.

Table 6.1. Common Phase I and Phase II biotransformations and the corresponding change in mass of the parent compound in high resolution MS

Biotransformation pathway

Formula

Mass change (amu)

Decarboxylation

—CO2

—43.9898

Deethylation

— C2H4

—28.0313

Oxidative dechlorination

+OH-Cl

— 17.9662

Demethylation

—ch2

— 14.0157

Desaturation

—2H

—2.0157

Oxidative defluorination

+OH-F

— 1.9957

Oxidative deamination

+O-NH3

— 1.0316

Double bond reduction

+2H

2.0157

Oxidation and desaturation

+O-2H

13.9793

Methylation

+CH2

14.0157

Hydroxylation, oxide

+O

15.9949

Hydrolysis

+H2O

18.0106

Hydroxylation and methylation

+O+CH2

30.0106

Dihydrolylation

+2O

31.9898

Acetylation

+COCH3-H

42.0106

Glycine conjugation

+NHCH2CO2H-OH

58.0215

Sulfonate conjugation

+SO3

79.9568

Hydrolylation and sulfonation

+O+SO3

95.9517

Taurine conjugate

NHCH2CH2SO3H-OH

107.0041

N-acetylcysteine conjugate

SCH2CH(CO2H)

161.0147

(mercapturic acid)

nhcoch3-h

Glucuronide conjugate

+C6H8O6

176.0321

Glutathione (GSH) conjugate

+CioHi7O6N3S-2H

305.0682

Table 6.2. Common neutral loss masses from product ion scans

Mass change (amu)

Species lost

17.0265 18.0106 20.0062 26.0157 27.0109 27.9949, 28.0313 30.0106, 30.0470, 29.9980 32.0262 33.9877 35.9767, 37.9737 42.0106, 42.0470,

42.0344 43.0058, 43.0422 43.9898 46.0055 56.0626 60.0211 63.9619 79.9262, 81.9241 127.9123

NH3 H2O HF C2H2 HCN CO, c2h4 ch2o, c2h6, no

CH3OH

H35Cl, H37Cl ch2co, c3h6, c2h4n

NHCO, C2H5N CO2 HCO2H

C4H8 C2H4O2 SO2 H79Br, H81Br HI

6.3.2.1 The Odd Nitrogen Rule

The odd nitrogen rule is a handy tool in metabolite identification. If a compound has an even number of nitrogen atoms (or none), its molecular ion or fragment ions ([M + H]+ or [M — H]—) will have an odd-numbered mass. If a compound has an odd number of nitrogen atoms, its molecular ion will have an even-numbered mass (see Table 6.3). This rule holds true because a nitrogen atom makes three bonds and has an even atomic mass unit (amu). Carbon and oxygen atoms form an even number of bonds and have even amu values, while a hydrogen atom forms an odd number of bonds (one) and has an odd amu.

Table 6.3. The odd nitrogen rule

Ions with nitrogen atoms

Even-electron ions

Odd-electron ions

Even or none Odd

Odd-numbered mass Even-numbered mass

Even-numbered mass Odd-numbered mass

6.3.2.2 Derivatization Techniques

Derivatization is a technique used to modify the potential site of modification with a specific functional group (see Table 6.4). From the perspective of metabolite identification, ideally, derivatization should be a one-step process without purification that can be performed at the bench with limited synthetic chemistry skills.

Table 6.4. Common derivatization reactions used to aid in metabolite identification

Moiety Reaction Reagent

Carboxylic acid Esterification Anhydrous methanol +HCl or diazomethane (TMS diazomethane)

Aldehyde Reduction to alcohol LiAlH4 or NaBH4

N-Oxide Reduction TiCl3

Amine Acetylation Acetic anhydride

Phenol Acetylation Acetic anhydride

6.3.2.3 Hydrogen/Deuterium (H/D) Exchange H/D exchange is a reaction in which exchangeable hydrogen atoms in a compound (or a metabolite) are replaced with deuterium. This is done by performing the reaction/incubation in D2O instead of H2O. D2O does not need to be 100% pure but the percent incorporation is calculated based on %D2O. Changes in molecular ions and fragment ions that contain exchanged hydrogen atoms are detected by MS. Note that if the samples are run in a mobile phase with H2O, exchangeable deuterium atoms will most likely be replaced back to hydrogen. To avoid this, a direct infusion can be carried out or D2O can be used in the mobile phase.

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