Back to the Top
The following message was posted to: PharmPK
Hi Everybody
I am using a d8 isotope for the quantitation of an analyte . The
column is PVA silica(4.0x50 mm) and the mobile phase is non aqueous
solvent combination with formic acid and ammonium hydroxide. I am
getting a separation of 0.3 min between the two peaks( deuterated vs
non deuterated) which is not normal. I did have confirmed the
structure of the d8 analogue by MS and NMR and the pattern is very
much matching with the non deuterated one.
I was wondering if anyone has faced the same problem. Is it really
possible to get this type of separation with a silica column.
Any quick input in this regard will be greatly appreciated.
Thanks for your time.
Bajpai
(Lakshmikant Bajpai)
Back to the Top
The following message was posted to: PharmPK
This phenomenon is due to the shorter C-D bond and, therefore, the
smaller molecular volume occupied by the d8 analog of your analyte.
The amount of separation is proportional to relative size of the
labeled moiety to the unlabeled compound. If, for instance, you are
analyzing d8 chlorotoluene vs. chlorotoluene, the relative size of
the d8-chlorotoluene would be smaller than the unlabeled
chlortoluene. If the d8 moiety were part of a molecule with a much
greater overall molecular weight, the d8 labeled compound might still
show some separation, though not as much separation as you are
currently witnessing. Of course, MS scientists like to co-elute the
labeled and unlabeled moieties so that they are sure of similar
extraction profiles for the two compounds and an analytical
distinction can be made based on mass difference alone.
Ian
Back to the Top
The following message was posted to: PharmPK
Hi Ian
Thanks for your input. The MW of compound is 447 which is not very
less. I myself has used a d7 IS for an analyte with MW 539 and got no
separation there, which was bothering me the most in this case.
Bajpai
(Lakshmikant Bajpai)
Back to the Top
The following message was posted to: PharmPK
Dear Ian,
Have you got any references for separation based on molecular volume for
deuterated compounds?
TIA
Frederik
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.at.auckland.ac.nz
Back to the Top
The following message was posted to: PharmPK
Dear Bajpai,
Initially, it may indeed seem "not normal" that you get separation
between d8- and d0-analogues. As Ian pointed out molecular size has got
something to do with this. However, there is (a lot) more to it: the
position of the labels relative to other chemical groups is important
too.
I think the ball got rolling on this with the introduction of the ICAT
technology. Below are some references that may be useful.
Interestingly, so-called stable isotope internal standards can also
cause ion suppression of the non-labelled analyte (LC-MS) and vice versa
(Liang et al.).
Rapid Commun. Mass Spectrom. 2003; 17: 2815-2821. Ionization enhancement
in atmospheric pressure chemical ionization and suppression in
electrospray ionization between target drugs and stable-isotopelabeled
internal standards in quantitative liquid chromatography/tandem mass
spectrometry. H. R. Liang, R. L. Foltz, M. Meng and P. Bennett
JOURNAL OF BIOSCIENCE AND BIOENGINEERING. Vol. 99, No. 1, 75-77. 2005.
An Isotope Effect on the Comparative Quantification of Flavonoids by
Means of Methylation-Based Stable Isotope Dilution Coupled with
Capillary Liquid Chromatography/Mass Spectrometry. EI'ICHIRO FUKUSAKI,
KAZUO HARADA, TAKESHI BAMBA, AND AKIO KOBAYASHI
Anal. Chem.2002, 74,3662-3669. Controlling Deuterium Isotope Effects in
Comparative Proteomics. Roujian Zhang, Cathy S. Sioma, Robert A.
Thompson, Li Xiong, and Fred E. Regnier
Kind regards,
Frederik Pruijn
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.at.auckland.ac.nz
Back to the Top
The following message was posted to: PharmPK
hi just saw this on deuterium substituted isotopomers.
we found the we could almost baseline resolve lipid isotopomers (d6/
d0), on a reveresed phase system. deuterum
subsitution definately changes the partitioning properties of
compounds, especially when in olefinic positions. it is
probably not enough to worry about biologically, but certainly
detectable through changes in retention character on
chromatographic systems (the deuterium compounds elute earlier. the
explaination probably has something to do with
the zero point energy of the molecule, ie deuteriums vibrate slower).
i also worked on a problem where deterium isotopomers cause mass
shifts in the d0 compound by ion trap analysis.
it was a few years back, and only gets bad at higher enrichments. it
was 'dueker et al. anal chem, title: ion
trap.......'
Stephen Dueker
President
Vitalea Science
www.vitaleascience.com
Provider of microdosing and Accelerator Mass Spectrometry solution
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)