Drug Metabolism in Drug Design and Development Basic Concepts and Practice

integration of knowledge-based metabolic prediction with data-dependent
LC/MSn to study the biotransformation of indinavir (Anari et al., 2004).
Potential metabolic pathways of indinavir were predicted from MDLI
metabolite database, which included two hydrolytic, two N-dealkylation, three
N-glucuronidation, one N-methylation, and several aromatic and aliphatic
oxidation pathways (Scheme 11.4). Them/zvalues of potential metabolites
resulting from a single or multiple common metabolic pathways were calculated.
Thism/zlist was then used to set up an inclusion list-dependent LC/MSn
instrument method. This approach led to the identification of 18 metabolites of
indinavir following incubation of the drug with human hepatic S9 fractions. The
inclusion list-specific data-dependent analysis for potential metabolites was
found to be effective in triggering MSndata acquisition for the corresponding
metabolites even though the signal intensities of these metabolites were very low.

11.5.2 Mass Defect Filter (MDF)

Mass defect is defined as the difference between the exact molecular weight and
nominal molecular weight of an element. The atomic mass scale defines
carbon-12 with a mass of exactly 12.0000 Da, therefore all other elements will
have a uniquely different mass defect. For example, the mass defects of
hydrogen and oxygen are 0.007825 and0.005085 Da, respectively. Therefore,
oxidation will introduce a mass defect of5.1 mDa, while glucuronidation will
introduce a mass defect of +32 mDa. The mass defects of common
nonsynthetic (Phase I) and synthetic (Phase II) metabolites typically fall
within 50 mDa. Therefore with LC/MS instruments capable of high mass
accuracy, it is possible to filter out matrix-related interference ions whose mass

N

N

N

N

H

OH

O

OH

O NH

N-Oxidation

N-Methylation

N-Glucuronidation

Heteroaromatic
hydroxylation

N-Glucuronidation

N-Oxidation

N-Dealkylation

Aliphatic oxidation

Alicyclic oxidation

Aliphatic oxidation

Amide hydrolysis

Aromatic hydroxylation

Benzylic hydroxylation

Aromatic and alicyclic

Hydroxylation

N-Dealkylation

SCHEME 11.4 Knowledge-based metabolic prediction of indinavir. Adapted from
Anari et al. (2004) with permission of the American Chemical Society.

346 APPLICATION OF LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY