Drug Metabolism in Drug Design and Development Basic Concepts and Practice

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metabolites are retained somewhat and not eluted in the HPLC solvent front.
Since feces displayed the most significant matrix effect among the samples
tested, it is recommended that injections contain the smallest possible amount
of fecal sample. Furthermore, when dealing with radioisotopes other than the


(^14) Cor biological matrices different from those tested in that study, potential
matrix effects of the samples on TopCount performance should be evaluated.
10.3.1.4 Radioactivity Recovery Determination Determination of the radio-
activity recovery from an HPLC analysis is a necessary procedure to ensure
accurate determination of all radioactive components in the original sample. In
addition, because metabolite profiling by MSC requires the additional step of
in vacuo removal of HPLC solvents, it is also important to determine whether
volatile metabolites are lost in the process. We have developed a simple method
to determine HPLC column recovery, plate recovery, and the total recovery for
an HPLC–MSC analysis (Zhu et al., 2005b). In general, aliquots of a sample
are injected onto an HPLC with and without an HPLC column. All effluent
from each HPLC run are separately collected and aliquots are analyzed for
total radioactivity by LSC with and without solvent evaporation. The DPM
values obtained are used to calculate the recoveries.


10.3.2 Stop-Flow HPLC-RFD


To improve the detection sensitivity of RFD, a stop-flow liquid radio-
chromatographic detection technique (Accurate radioisotope counting, ARC)
was recently developed by AIM Research Company. The stop-flow RFD
system offers three operation modes: (1) by-fraction; (2) by-level; and (3)
nonstop (Nassar et al., 2003, 2004). The by-fraction mode performs stop flow
in preset count zones or an entire HPLC run. The by-level mode performs stop
flow when radioactivity is above a preset minimal value. The nonstop mode
operates the same way as the regular RFD. Figure 10.5 displays typical
radiochromatograms acquired by the three operation modes on a stop-flow
RFD instrument after a radioactivity sample was repeatedly injected. The data
showed that the by-fraction analysis at the 1 min counting time was
approximately 10-fold more sensitive than traditional RFD. On the contrary,
the time to complete a radiochromatographic run using by fraction mode was
increased to 4 h from 45 min in the nonstop flow analysis. The by level mode
provided a shorter total run time (52 min) than the by-fraction mode and
significantly better sensitivity than the nonstop mode.
The stop-flow technique greatly extends the capability of RFD by improving
its sensitivity, while it retains the advantages of online radiodetection. The
stop-flow RFD method has the flexibility to allow it to operate in different
modes suitable for high speed analysis, automation, and higher sensitivity. The
new online radiochromatographic technique has been employed in profiling of
in vitroandin vivometabolites and metabolite structural characterization by
coupling with a mass spectrometer (Nassar et al., 2003, 2004). Stop-flow RFD


298 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES

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