Biomedical Engineering Reference
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Fig. 7 Chemical structures of 5a -androstan-17 b-ol-3-one-2, 2, 4, 4-D 4 ( a ) and 5a -androstan-
17b-ol-3-one-16, 16, 17-D 3 ( b ) and schematic representation showing how deuterium-hydrogen
exchange occurs during keto- and enol- form conversions ( c )
analyte (carvedilol-S at the retention time of 1.93 min) than for the corresponding
internal standard (at the retention time of 1.91 min) with a difference of about 25 %.
This type of differential ion suppression was also believed to be the cause for the
significant differences between original and reinjection concentrations observed for
run reinjection within validated postpreparative stability on a different LC-MS
instrument, though all calibration standards and quality control samples were
accepted in both runs [ 36 ] .
Another issue with deuterated internal standards is the possibility of deuterium
and hydrogen exchange in sample matrix or solvent. For example, [ 13 CD 3 ]rofecoxib
was shown to be isotopically unstable in plasma and water containing solvent and
an efficient exchange between deuterium and hydrogen prevented its use as the
internal standard in an LC-MS method. On the other hand, the isotopic integrity of
[ 13 C 7 ]rofecoxib was maintained [ 37 ]. Moreover, the positions of deuterium atoms
can also have an impact on the stability of a deuterated internal standard. As shown
in Fig. 7 , when deuterium atoms are adjacent to a keto group (5a -androstan-17 b -ol-
3-one-2, 2, 4, 4-D 4 ), a significant loss of deuterium atoms was observed at pH 6 due
to potential conversions between keto- and enol- forms [ 38 ] . While the deuterium
atoms are far away from the keto- group, such as 5a -androstan-17 b -ol-3-one-16,
16, 17-D 3 , no such deuterium-hydrogen exchange was observed.
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