analyzed by a noncompartmental method (Gibaldi and Perrier, 1982). The peak
plasma concentration,Cmax, and the time to reach peak concentration as the first
occurrence,Tmax, were recorded directly from experimental observations. The
area under the plasma concentration versus time curve (AUC) was calculated by
a combination of the trapezoidal methods. The AUC was calculated from time
zero to the time,T, of last measurable concentration [AUC(0–T)]. The first-order
rate constant of decline of radioactivity concentrations and unchanged
muraglitazar, expressed as equivalents of muraglitazar, in the terminal phase
of each plasma concentration versus time profile,K, was estimated by log-linear
regression (using no weighting factor) of at least three data points that yielded a
minimum mean square error. The absolute value ofKwas used to estimate the
apparent terminal elimination half-life,t1/2.
18.3.4 Metabolite Profiling
Various samples (plasma, urine, bile, fecal extracts) are analyzed by HPLC to
determine metabolite profiles. In general, HPLC is performed on a system
equipped with two pumps, an auto-injector, and a diode array detector. A
reverse-phased HPLC column is normally used for metabolite separation. A
gradient of two solvents, A and B, is usually used with Solvent A as aqueous
and Solvent B as organic. For metabolite profiling ofin vivosamples, usually a
60 min HPLC run is needed to ensure good separations of all metabolites.
Recovery of radioactivity after HPLC column needs to be checked to ensure
that all radioactive material is eluted from the column. For detection of
radioactivity, HPLC fractions may be collected and radioactivity is deter-
mined. To ensure the accuracy of the results, it is important to check the
recovery of radioactivity against the known amount of radioactivity injected.
Loss of radioactivity could be due to retaining in the HPLC column, injector
misfunction, volatile metabolites, or quenching by matrices. The limit of
quantification is 15 disintegrations per minute (DPM) for microplate
scintillation counting (MSC). Alternatively, HPLC is coupled to a flow
radiochemical detector (e.g.,b-RAM, IN/US systems, Tampa, FL). A liquid
cell usingb-RAM instrument with 1 mL/min HPLC flow and 2 mL/min
cocktail flow (INUS 2:1) with a 500–600mL cell is recommended (for a balance
of peak resolution and counting time). It is necessary to calibrate the cocktail
pump and check counting efficiency of theb-RAM. Under these conditions,
the counting efficiency is 80% for^14 C and is 30% for^3 H, and the
quantitation limit is700 counts per minute (CPM) per HPLC peak for^14 C.
The decision on whether to use online or off-line radioactivity detection should
be based on the amount of radioactivity in a sample and the desired detection
limit of analytes. For example, in order to detect a metabolite that accounts for
1% of total radioactivity in a sample (^14 C), it requires approximately 1500
DPM per injection for HPLC–MSC and70,000 CPM for HPLC–b-RAM.
Biotransformation profiles are prepared by plotting the CPM or DPM values
against time-after-injection. Radioactive peaks in the biotransformation
SAMPLE ANALYSIS 583