Biomedical Engineering Reference
In-Depth Information
about 12 min or even less, to reach an excellent overall resolution for all considered
metabolites including (
E
/
Z
) endoxifen isomers and position isomers of 4-hydroxy
and 4-hydroxy-
N
-desmethyl-tamoxifen. Alternatively, Zweigenbaum J and Henion
J [
235
] developed a high-throughput analysis technique for the separation of tamox-
ifen, 4-hydroxytamoxifen, and other SERMs within only 30 s using a narrow-bore
short analytical column packed with small (3 mm) particles. Separation was
performed under isocratic conditions at flow rate of 500 ml/min. Gjerde et al. [
238
]
also described an online SPE-LC-MS/MS procedure where chromatographic reso-
lution of tamoxifen and five of its metabolites was achieved within 6 min using a
monolithic silica column (Separation was performed under a gradient program at a
flow rate of 500 ml/min). However this method, like other HPLC assays, clearly
failed to resolve all the hydroxylated and
N
-desmethyl-hydroxylated tamoxifen
metabolites.
Tamoxifen is metabolized to a plethora of
N
-desmethylated, hydroxylated, and
their corresponding glucurono- or sulfo-conjugated metabolites. Some of these
hydroxylated metabolites are position isomers (such as 4-hydroxy-tamoxifen,
3-hydroxy-tamoxifen, and 4¢-hydroxy-tamoxifen; endoxifen; and 4¢ -hydroxy-
N
-
desmethyl-tamoxifen) and have similar molecular mass and fragmentation pattern
(Table
4
). Besides,
E
/
Z
isomerization (around the ethylenic double bond of tamox-
ifen and its metabolites) may occur either in biological samples or as contaminants
or degradation products in pure standards. Some pure standards are also best syn-
thesized as an equimolar
E
/
Z
mixture. Therefore, the chromatographic resolution
of these metabolites and their (
E
/
Z
) geometric isomers is of paramount importance
to ensure reliable and accurate bioanalytical methods.
However, of the LC-MS and LC-MS/MS methods developed so far for the com-
prehensive and quantitative study of levels variability in tamoxifen metabolites,
there is limited data with respect to the resolution of both 4-hydroxytamoxifen and
endoxifen position isomers (notably 4¢-hydroxylated metabolites) and their corre-
sponding (
E
/
Z
) geometric isomers. In fact, apart from the most recently published
articles [
145,
173,
196,
245-
247
], no data have been provided regarding this issue.
We were the first group that focused on method selectivity and on the effective sepa-
ration on potentially interfering hydroxylated tamoxifen metabolites. This has
allowed us to identify for the first time the occurrence of 4¢ -hydroxy-tamoxifen and
4¢ -hydroxy-
N
-desmethyl-tamoxifen and to estimate their plasma levels in a subset
of BC patients [
145
]. Such differences in chromatographic performances, between
assays, can thus affect the selectivity, the accuracy, and the reliability of some of the
proposed bioanalytical methods, potentially leading to discrepant data (or results)
between the PK, PG-PK and PK-PD studies. Actually, Mürdter et al. [
196
] reported
twice or even higher differences in median concentrations of (
Z
)-endoxifen between
studies conducted in the United States, Japan and Norway. They also found a plau-
sible explanation for these discrepancies in method selectivity problems. Madlensky
et al. [
173
] compared the performance of their assay to that of another laboratory
performing similar measurements of tamoxifen metabolites in human serum. They
found discordant results for 4-hydroxy-tamoxifen levels measured in the same
serum samples.
