2110 A. D. N. Vaz et al.
cryopreserved hepatocytes were obtained commercially
from Gentest Laboratories and Celsis In Vitro Technologies.
For proprietary compounds examined in this study, only the
functional components relevant to the study are shown, with
an ’R’ representing the rest of the molecule.
stopped with a 5ꢀ volume of acetonitrile and processed in
the same manner as the diclofenac reaction.
Reaction with hydroxylamine
An aliquot (50–100 mL) of the re-dissolved extracts from
microsomal or hepatocyte reactions, urine, or plasma was
treated with an equal volume of a 55% aqueous hydro-
xylamine solution. After standing at room temperature for
1 to 12 h an aliquot was analyzed by liquid chromatography/
tandem mass spectrometry (LC/MS/MS). With the diclofe-
nac acyl-glucuronides, the reaction was judged to be
complete after 2 h at room temperature based on the absence
of glucuronides in the reaction mixture as determined by
mass spectrometry.
Lasofoxifene glucuronides
In the development of lasofoxifene, definitive metabolic
studies using radiolabeled drug showed several urinary
phenolic glucuronides.23 Mouse urine from a 14C-labeled
lasofoxifene mass balance study was used to examine the
reactivity with hydroxylamine.
N-Carbamoyl-glucuronides of desipramine and
duloxetine
The carbamoyl-glucuronides were synthesized as follows.
Desipramine (200 mM) or duloxetine (200 mM) was incubated
with dog liver microsomes (2 mg/mL) in 100 mM sodium
bicarbonate buffer adjusted to pH 7.5 with hydrochloric acid
and 0.2% bovine serum albumin, supplemented with uridine
diphosphoglucuronic acid (UDPGA, 5 mM) and alamethicin
(50 mg/mL). The reactions were maintained at 378C and
flushed continuously with carbon dioxide. After 120 min the
incubation was supplemented with additional UDPGA
(5 mM), and after 240 min a 4ꢀ volume of acetonitrile was
added to the reaction to precipitate protein. The supernatant,
after centrifugation at 1100 g for 5 min, was evaporated to
dryness in a vacuum centrifuge. The residue was re-
dissolved in 0.1% formic acid/acetonitrile (90:10) (3 mL)
and the supernatant used to conduct the derivatization
reaction without purification.
LC/MS/MS
Multiple ThermoFinnigan mass spectrometric platforms
integrated with either Surveyor or Agilent liquid chromato-
graphic systems were used for analysis, and include TSQ
Quantum triple quadrupole, LCQ-Deca and LTQ ion trap
instruments. In each case the mass spectrometer was
optimized for the parent of the particular compound under
study. Chromatographic separation was generally done on
C-18 reversed-phase columns using gradients of acetonitrile
with ammonium formate/acetate in the pH range from 3.0 to
6.8. The exact condition used in each analysis is indicated
in the related figure legends. As multiple systems and
analytical columns were used in this report, the identity of
substrates, metabolites, and derivatives is based entirely on
their mass spectral fragmentation patterns and not on their
chromatographic retention times.
N-Glucuronide of compound Y
RESULTS AND DISCUSSION
This metabolite was identified in rat bile from a study with
14C-compound Y in bile-duct cannulated rats. Its identity as
an N-glucuronide at the indole nitrogen was established
from its fragmentation pattern and exact mass on a
ThermoFinnigan Orbitrap mass spectrometer.
Synthesis and reactivity of diclofenac-
glucuronides
Figure 1(a) shows the UV and the ion current chromatograms
at m/z 296 and 472 for the reaction of diclofenac with rat liver
microsomes supplemented with UDPGA and alamethicin.
Diclofenac eluted at 36.9 min (UV). The extracted ion
chromatogram for the glucuronides (m/z 472) of diclofenac
shows four chromatographically resolved peaks eluting at
29.8, 31.0, 31.7 and 32.0 min (UV). The MS1 profile for the
peak at 29.8 min shows an ion current at m/z 296, which is
also the most intense ion current in the MS2 spectrum
(Fig. 1(b)) and corresponds to a loss of 176 Da, which is
characteristic of glucuronides. This suggests considerable in-
source fragmentation for this glucuronide of diclofenac. The
fragmentation patterns of the peaks at 31.0, 31.7 and 32.0 min
are similar, with no apparent in-source fragmentation (m/z
296 in the MS1 chromatograms of Figs. 1(c), 1(d), and 1(e)).
Their MS2 spectra show losses of 18 and 194 Da as most
intense (Figs. 1(c), 1(d), and 1(e)) and loss of 176 Da being
significantly less intense, suggesting these glucuronides are
likely to be rearrangement products of conjugated diclofenac
due to intramolecular acyl group migration. Similar
observations have been made for the ion source stability
of the acyl-glucuronides of muraglitazar.24 After treatment of
the reaction extract with hydroxylamine the m/z 472
Biochemical synthesis of diclofenac acyl-
glucuronides
Diclofenac (100 mM) was incubated at 378C with rat liver
microsomes (1 mg/mL) in 50mM potassium phosphate
buffer pH 7.4, supplemented with UDPGA (1 mg/mL)
and alamethicin (50mg/mL). After 60 min a 5ꢀ volume of
acetonitrile was added to the reaction to precipitate protein.
The supernatant, after centrifugation at 1100 g for 15min, was
evaporated to dryness in a vacuum centrifuge. The residue
was re-dissolved in DMSO/acetonitrile/water (20:10:70,
300 mL), centrifuged to remove insoluble matter, and the
supernatant used to either purify the acyl-glucuronide(s) or
conduct derivatization reactions without purification.
Glucuronides of carbazeran and compound X
Carbazeran or compound X (10 mM) was individually
incubated at 378C with cryo-preserved human hepatocytes
(750 000 cells/mL) suspended in Williams E media and
gassed with 5% CO2/95% O2. After 4 h the reactions were
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 2109–2121
DOI: 10.1002/rcm
Vaz, Alfin D. N.
