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Dear All,
I am getting two different retention times for deuterated internal standard (1.4 min) and Analyte
(3.5 min) using Waters Acquity HILIC column. Ideally, IS and analyte have same physicochemical
properties except their molecular weights so both of these should elute at same retention time.
Expert opinion is requested.
Regards,
Abhisheak
Abhisheak Sharma, Ph.D.
Post-doctoral Research Associate
Department of Pharmaceutics & Drug Delivery
The University of Mississippi
101 Faser Hall,
University, MS 38677, United States (US)
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From: Dario Doller
Do you have a H-1 and C-13 NMR spectra of the deuterated compound? How do they compare with the
protonated version of your compound?
Did you make the deuterated compound from the protio-version as the starting material?
Dario
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From: Beumer, Jan H
Dear abisheak,
It is not uncommon to see slightly disparate retention times, but 2 min is much. The heavier
isotopes result in shorter binding distances and slight differences in polarity.
I suggest you confirm identities of analyte and IS, and eg compare product ion spectra.
Jan
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From: Clay Frederick
Whatever is eluting at 1.4 min is chemically (not isotopically) different from whatever is eluting
at 3.5 min ...
Regards,
Clay
Clay B. Frederick, PhD, DABT, ATS
CBF Safety Assessment Consulting, LLC
1621 Kenmare Dr.
Dresher, PA 19025
Website: www.cbfsaconsulting.com
Email: clayfrederick.-at-.cbfsaconsulting.com
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Dear Sharma,
I think it is better to check the integrity of the labelled standard it may have been degraded
during derivitization.
Dr Zafar Iqbal.
Meritorious Professor
Department of Pharmacy
University of Peshawar
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Hi Abhishek,
Please reconfirm MRM for drug and IS
For example if your drug MRM is 100/50 and if IS is d4 than MRM of IS should be 104/54 or 104/50.
Thanks
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From: Edward O'Connor
How deuterated is the IS?
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From: Abhisheak sharma
Thanks! every one for valuable suggestions. I did NMR of the deuterated compound and find that it
was not what it supposed to be. It was wrongly supplied by the manufacturer.
Regards,
Abhisheak
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Hi, Abhisheak
The "isotope effects" can cause the analyt and deuterium labeled IS to be separated on the HPLC
column, especially the deuterium atom is close to the hydroxyl and carbonyl group in the compound.
To solve this problem, you can optimize the HLPC condition to get same retention time, or select 14C
labeled compound as internal standard because 14C usually causes less isotope effects.
Xiaoming
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The retention time difference is far too large to be attributed to any sort of isotope effect. From
my perspective, there is a substantive chemical structure difference between the two analytes that
provides a significant difference in the interaction with the column backing (i.e. providing
chromatographic separation of the analytes) ...
Regards,
Clay
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I believe he described that a NMR run on the label and native material showed that they were in fact
two different compounds
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Yes, it is true. We received a different deuterated compound. It has same substitution at different
nitrogen than the analyte. Both were not identical therefore retention time was different.
Thanks,
Abhi
Abhisheak Sharma, Ph.D.
Post-doctoral Research Associate
Department of Pharmaceutics & Drug Delivery
The University of Mississippi
101 Faser Hall,
University, MS 38677, United States (US)
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