CYPs involved in the metabolism, one has a high affinity and low capacity
(Kappm1¼ 0 : 46 mM;Vappmax1¼ 17 :5 nmol=min=mg protein), and the other has a low
affinity and high capacity (Kappm2¼ 24 : 5 mM;Vappmax2¼ 65 :1 nmol=min=mg protein).
The basis for these differences has not been resolved.
13.4 QUANTITATIVE ANALYTICAL METHODS
Accurate data acquisition is essential for successful metabolic studies. The
current trend from HPLC-based to LC/MS/MS-based analytical tools in
ADME research, particularly in the pharmaceutical industry, demonstrates
this requirement for accurate data acquisition. General aspects of the operation
of LC/MS/MS are described below, together with a brief overview of
traditional HPLC systems.
13.4.1 HPLC/UV/FL
These systems, traditionally used for the quantitative determination of
metabolites and/or parent compounds, require the compounds to exhibit UV
absorbance and/or FL emission, as well as to be absolutely resolved
chromatographically. The use of the extended run times necessary to achieve
requisite resolution is counterproductive for rapid-throughput analysis.
Aromatic compounds are readily analyzed using a HPLC/UV system because
of their chromophores with high molar absorptivity, typically eliciting UV
absorbance between 240 and 260 nm (single rings), 270 and 290 nm (two fused
rings particularly for the heterocyclic systems) and at more than 300 nm
(multiple conjugated ring systems). Moreover, compounds that exhibit strong
UV absorbance can potentially be monitored by fluorescence. The fluorescent
emission wavelengths are longer than the excitation wavelengths, due to the
lower emission energy, often by at least 25 nm. For example, if a compound
absorbs UV at 320 nm, it is expected to emit fluorescence at wavelengths
between 350 and 400 nm, as occurs for the fluorescent warfarin metabolites,
6- and 7-hydroxywarfrin (Takahashi et al., 1997).
Conversely, the HPLC/UV systems have limited applicability for monitor-
ing aliphatic compounds without conjugatedpsystems due to the rather poor
UV absorbance, and an alternative tool, LC/MS/MS in particular, is
recommended if available.
13.4.2 LC/MS/MS
13.4.2.1 System and Principle LC/MS/MS provides a standard analytic
system for DMPK studies in the pharmaceutical industry. This system is
versatile, and does not have the prerequisites of LC/UV/FL systems. Instead,
MS ionization and ion fragmentation of the molecules of interest become
432 DETERMINATION OF METABOLIC RATES AND ENZYME KINETICS