radiometric HPLC detector or fraction collection and liquid scintillation
counting can be employed. The latter method is more sensitive especially for
tritiated compounds.
Direct HPLC assays for glucuronide production can be easily done on
reversed-phase columns if an authentic standard of the glucuronide is available,
however, for many drug candidates this may not be possible. Since most
glucuronides have identical absorbance spectra as their aglycones (the sugar does
not contribute to absorption at wavelengths above 210 nm), one can construct a
standard curve with the aglycone for quantitation of the glucuronide, assuming
there are no matrix interferences. To insure that the product is a glucuronide, it is
usually wise to collect the peak and treat with beta-glucuronidase to ensure that
the aglycone peak is observed after hydrolysis. Alternatively, glucuronidase
treatment of an incubation (lacking saccharo-1,4-lactone) with subsequent loss
of the glucuronide peak is sufficient evidence. However, someN-glucuronides are
often resistant to beta-glucuronidase hydrolysis, for example, lamotrigine (Sinz,
1991) and olanzapine (Kassahun, 1997). Primary and secondary amino
glucuronides are generally easily cleaved with dilute acid, whereas acyl (ester)
glucuronides are easily cleaved with dilute base (0.1 N NaOH and mild
heating).
LC–MS methods on either single quadrupole or time-of-flight instruments or
tandem mass spectrometers are sensitive, and efficient procedures to quantitate
glucuronide production if an authentic standard is available. Glucuronides
readily fragment with a neutral loss of 176 amu to produce the aglycone
fragmentation product. Glucuronides have a pKaof around 4, and thus are
readily detected under negative ion conditions at pH 4.5 or higher by electrospray
or assisted electrospray, so ammonium acetate buffers are commonly employed.
Trifluoroacetic acid can cause ion suppression especially in negative ion mode,
but may improve glucuronide retention on C8 or C18 columns by protonating
the carboxylic acid group. Ion pairing of charged carboxyl groups with
trieethylamine or morpholine can be used to improve retention. If the drug
substrate is a weak base, positive ion mode may also be used.
3.1.7 Enzyme Selective Substrates and Inhibitors
Enzyme selective substrates and inhibitors have been widely used to distinguish
individual cytochrome P450 activities, but this has been problematic for
glucuronidation because of the overlapping substrate specificity for some of
these enzymes. This is especially true for the closely homologous enzymes
UGT1A3 and UGT1A4, and UGT1A7–UGT1A10. However, selective
substrates for some of the enzymes have been well characterized.
UGT1A3 and UGT1A4 are>95% homologous and share many common
substrates especially tertiary amine substrates. There are some substrate
differences. The general UGT substrate 4-methylumbelliferone is a substrate
for 1A3 but not 1A4. Bile acid conjugation at the 24-COOH group and
fulvestrant are much more efficiently catalyzed by 1A3 versus 1A4, whereas
UDP-GLUCURONOSYLTRANSFERASES 59