Drug Metabolism in Drug Design and Development Basic Concepts and Practice

An example of using stop-flow HPLC–RFD to determine the enzyme
kinetic parameters of the glucuronidation of muraglitazar in human UGT1A3
was recently presented (Zhao, 2004). Muraglitazar was incubated with
recombinant human UGT2B7 enzyme and UDPGA. These samples were
analyzed using the stop by-fraction mode so that the HPLC flow rate was
stopped and counted for 1 min in the selected time zones when significant drug-
related components passed through the RFD cell (Fig. 10.11). The relative
distribution of the muraglitazar glucuronide in the sample was determined and
its concentrations in the incubations were calculated by multiplying the relative
distribution to the initial drug concentration in the incubations. The precision
and accuracy of the stop by fraction analysis for determining the percentage
distribution of the drug and its glucuronide were found to be quite good
(Table 10.3). Accordingly, the relationship of muraglitazar glucuronidation

1 2

3

4

CPM

1 2

3

4

0.00 5.00 10.00 15.00 20.00 25.00

-0.1

0.4

0.9

1.4

1.9

Retention time (min)

0 20 40 60

0

200

400

600

800

1000

1200

1400

Muraglitazar

Glucuronide of muraglitazar

Time (min)

Glucuronide of

muraglitazar

Muraglitazar

FIGURE 10.11 Quantification of muraglitazar glucuronide in the incubation with
UGT1A3 by off-line HPLC-LSC (four fractions per min) (top panel) and online stop-
flow HPLC–RFD (operated with the stop by-fraction mode) (bottom panel).

APPLICATION OF NEW RADIOCHROMATOGRAPHY TECHNIQUES 311