362
because the metabolites can be charged or uncharged, they
can be thermally unstable, and derivatization is unnecessary.
Normal phase columns (silica gel) can be used for uncharged
metabolites, and reversed phase columns (silica gel to which
C4 to C18 alkyl chains or any of a variety of more exotic
lipophilic moieties are attached to give a hydrophobic envi-
ronment) can be used for neutral or charged metabolites.
For GC separation, the metabolites must be volatilized. This
often requires prior derivatization[33] in order for the metab-
olites to volatilize at lower temperatures. Carboxylic acids
can be converted into the corresponding methyl esters with
diazomethane; hydroxyl groups can be trimethylsilylated
with bis-trimethylsilylacetamide or trimethylsilylimidazole
in pyridine. Ketone carbonyls can be converted into O-sub-
stituted oximes. With radiolabeled compounds, the radio-
activity can be monitored and separated directly from the
HPLC column using an in-line radioactivity detector.
8.3.3. Identification
The two principal methods of metabolite structure
identification are mass spectrometry and NMR spectrom-
etry. It is preferable to link the separation and identification
steps by running tandem LC-MS, tandem GC-MS, or tandem
CE-MS. These methods are sufficiently sensitive to identify
subnanogram amounts of material. The most popular method-
ology is tandem LC-electrospray ionization mass spectrom-
etry by which a metabolite extract (or urine directly) can be
injected into the HPLC, and each peak run directly into the
mass spectrometer.[34] Similarly, tandem CE-electrospray
ionization mass spectrometry has become a very valuable
tool for separation and identification of biomolecules and
drug metabolites.[35] In LC-tandem mass spectrometry/mass
spectrometry (LC/MS/MS), the HPLC is connected to a mass
spectrometer that is capable of not only obtaining parent ion
data but also conducting fragmentation of the parent ion and
obtaining product ion data. This technique can rapidly pro-
vide both a full mass spectrum (MS) and a product ion mass
spectrum (MS/MS) for each metabolite.[36] Ultrafast gradient
HPLC-MS/MS can produce run times of less than 5 min.[37]
 
In this way there is less chance for metabolite degradation or
loss, and workup procedures for mass spectrometry sample
preparation are eliminated. Mass spectrometric properties
are determined using different ionization techniques. Com-
mon vacuum ionization sources include electron impact (EI),
chemical ionization (CI), matrix-assisted laser desorption/
ionization (MALDI), fast atom bombardment (FAB), and
secondary-ion mass spectrometry (SIMS). The development
of HPLC coupled to atmospheric pressure ionization sources,
namely, electrospray ionization (ESI) and atmospheric pres-
sure chemical ionization (APCI) mass spectrometry, have
transformed the status of drug metabolism studies from their
former minor role in drug discovery to their current impor-
tant function during the drug discovery process. These latter
The Organic Chemistry of Drug Design and Drug Action
LC/MS/MS methods are used not only for drug metabolism
studies but also to investigate drug pharmacokinetics (absorp-
tion, bioavailability, and clearance), where the focus is less
on identification of metabolites and more on quantification of
parent drug in the systemic circulation as a function of time
(see section on quantification below). The trend in the phar-
maceutical industry now is to initiate pharmacokinetic and
metabolism studies as early as possible in the drug discovery
process to aid in the selection of compounds that have the most
druglikeness and best chance for survival through the many
drug discovery and drug development hurdles to avoid late
attrition of drug candidates.[38] With these HPLC/atmospheric
pressure ionization mass spectrometric techniques, assess-
ment of in vivo plasma half-lives and metabolic degradation
can be made rapidly on a large number of drug candidates.
A brief description of different mass spectrometric tech-
niques follows. Electron impact mass spectrometry (EI-
MS) involves the bombardment of the vaporized metabolite
by high-energy electrons (0–100 eV), producing a molec-
 
ular radical cation (M+·) having a mass equivalent to the
molecular weight of the compound. The electron bombard-
ment causes bond fission and the positively charged frag-
ments produced are detected. The mass spectrum is a plot of
the percentage of relative abundance of each ion produced
vs the mass-to-charge ratio (m/z).
CI mass spectrometry is important when compounds
do not give spectra containing a molecular ion, generally
because the molecular ion decomposes to give fragment
ions. With CI-MS, a reagent gas such as ammonia, isobu-
tane, or methane is ionized in the mass spectrometer and
then ion–molecule reactions, such as protonation, occur
instead of electron–molecule reactions. This soft ionization
process results in little fragmentation. Fragment ions in this
case are almost always formed by loss of neutral molecules,
and as a result, much less structural information can be
gleaned relative to EI-MS.
A variety of mass spectral techniques for nonvolatile or
higher mass compounds, including peptides and proteins,
also is now available.[39] MALDI is a soft ionization tech-
nique that is important for analyzing biopolymers.[40] It
has the ability to produce gas-phase ions with little or no
molecular fragmentation. FAB ionization involves the bom-
bardment of a liquid film containing the nonvolatile sample
with a beam of energized atoms of xenon or argon. This
method is also useful for thermally unstable compounds.
SIMS is similar to FAB except that energetic ions (Xe+ and
Ar+) instead of atoms are used in SIMS.[41]
Two important atmospheric pressure ionization tech-
niques arose out of the need for an ionization source that
provided even softer ionization (less fragmentation of the
molecular ion) and as a convenient interface with a liquid
chromatograph. With ESI, ions are generated in solution
phase, then the carrier solvent is evaporated, and a gas-phase
ion is produced.[42] In contrast to ESI, APCI is a gas-phase