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Hi,
I request for suggestions from the forum for a problem in LC-MS/MS
method development.
I have been trying to extract a NCE using organic solvents and
various combinations of organic solvents.
I reconstituted the extracted samples using the mobile phase.
I observed peak splitting in the reconstituted samples and the
split peak is appearing with all the solvents I have tried.
Standard solutions of the compound in the mobile phase are not
giving any such split peak.
I took the solvent in a tube, evaporated the solvent and
reconstituted with a standard solution.
Surprisingly, I could see the split peak in the reconstituted sample.
It appears to me that the peak splitting is related to solvent
extraction.Does any body in the group faced this kind of problem?
I would appreciate any sugestions in this regard
Thanks & Regards,
Nageshwar.
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Dear Nageshwar,
Isn t the compound degrading in the solvent you are using? Have you
tried injecting an academic solution of your compound in several
solvents directly into the MS (so, without chromatography) and scan
for different M/Z to have an indication of stability of your compound
in several solvents.
Did you validate the extraction procedure? Aren t you extracting more
compounds with your extraction method besides the compound of your
interest?
Yours sincerely,
--
Rob ter Heine, MSc, PharmD
Department of Pharmacology, Slotervaart Hospital
Amsterdam, The Netherlands
E: aprth.-a-.slz.nl
T: 020-5124737
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The following message was posted to: PharmPK
Hi Nageshwar
Could your 'split peak' actually be a degradation product that is
partially resolved from your parent peak? Is your assay stability
indicating?
The fact that peaks resulting from injections of your standard
solution (in the same matrix as your extracted samples) are normal
makes this unlikely to be a chromatography problem i.e. a true split
peak. Have you investigated whether your compound is degrading under
your extraction conditions? If not, it might be worth trying some
forced degradation studies to perhaps provide a clue about possible
mechanisms of sample degradation.
Good luck!
All best wishes
Ann
Dr Ann Rigby-Jones
Anaesthesia Research Group, Peninsula Medical School, Universities of
Exeter and Plymouth, UK.
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The following message was posted to: PharmPK
Hi Ann and and Rob,
Thank you very much for ur response.
I would not think that the split peak is an impurity.
Because, Iam using MRM method which you all know that highly specific.
Moreover, I have injected the standard solution of the compound
reconstituted in the solvent dried tube and it is also giving the
split peak.
Best Regards,
Nageshwar.
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Consider:
Decreasing sample concentration, decreasing sample volume injection,
preparing the sample in the mobile phase composition.
Stan Alekman
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Hi Nageshwar
I think its related to your injection volume or the reconstituion
solution. Also check for the any dead volume in the line. First of
all check all the connections to be perfect and then make several
injections with different injection volume. If its still there
certainly its not the reconstitution solution or the injection volume.
In your case since its present in the std also so it may not be
impurity but you could see an impurity even in the MRM, even though
its very specific but again its just based on the molecular weight
and several molecules may have the same molecular weight( its a known
fact).
Hope it will help.
Bye
Bajpai
(Lakshmikant Bajpai)
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Hi, Nageshwar,
According to what you described, I think that it is most likely caused
by the elution mode (isocratic or gradient) you used. May I suggest you
to re-optimize your mobile phase and elution mode in your trouble
shooting? Hope it is helpful.
Good luck!
Zhi
Department of Pharmacology
National University of Singapore
Tel: (65)68745493
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Hi Nageshwar,
I assume that your extraction method is liquid- liquid extraction, as
you mentioned that even after spiking standard solution in the dried
residue, you were getting splitting.
I guess, organic solvent residue may cause this problem. Dry samples
with temp. around 40*C or after reconstitution centrifuge the samples.
Hope this will help.
Jignesh Kotecha
Torrent.
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The following message was posted to: PharmPK
Hello Nageshwar,
I had a similar problem with peak splitting in an HPLC which I
resolved by
using the mobile phase as my diluent for sample preparation. Any
chance of
there being an isomer (enatiomer/diastereomer) in your mixture?
James Ladd
Erimos Pharma
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Hi Nageshwar
Did you try to evporate the extraction solvent and then
reconstituting it with your mobile phase. Try the same with different
reconstitution solvents like methanol, acetonitrile etc or a
combination of both with and without water. It would be good idea to
check for the solvents that are being used for extraction purposes.
Hope this helps
Manish Issar, Ph.D
Sandoz (Formerly Eon Labs Inc.)
4700 Eon Drive
Wilson, NC 27893
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Peak splitting is usually caused due to instability in mobile phase
pH so I
suggest you adjust the pH or change the composition of buffer in your
mobile
phase it should solve the problem.
Prasad Tata, Ph.D.
Mallinckrodt, Inc.
Saint Louis, MO 63134
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The following message was posted to: PharmPK
I've been following comments of the group regarding the question of
"peak
splitting" raised by Mr.Nageshwar. I think, most of the comments were
"general" to "peak splitting" rather than being "specific" to his
situation.
I'll go into little bit basics of "peak splitting". There could be many
possible reasons for peak splitting such as:
1. Sample volume too large (although reconstituted in mobile phase)
2. Injection (reconstitution) solvent is too strong
3. Column void or channelling
4. Blocked column frit
5. Leaky injector port
What I understand from the information provided by Mr.Nageshwar is that:
None of the above are possible reasons for his peak splitting as he
did not
find split peak when he injected standard solution in mobile phase
(remember
that the evaporation step is not there for this) which was injected and
eluted under similar LC and MS conditions. Therefore it is clear that
the
problem is not with injection solvent, column, inj port or the MS.
Next comes the point where he did see peak splitting when he
evaporated some
organic solvent which was injected after reconstituting using standard
solution. This confirms that the problem is either in the organic
solvent or
evaporation procedure.
What I suggest to Mr.Nageshwar to do is:
1. Check whether the organic solvent he used is contaminated with some
compound that is eluting close to his analyte of interest.
2. If that is not the culprit, then it should be the contaminated
needles of
the evaporation setup that washes into the sample when not used
cautiously
(i.e., too close to the solvent in the tube or too high a gas flow that
causes solvent to splash onto the needle).
I could say with confidence that the possible reason could be the
"contaminated needles" as I have faced this problem my self once. In any
case, it is advisable to dismantle the needles and clean them by
sonicating
in some organic solvent every now and then.
I would like to hear from Mr.Nageshwar with his findings.
Cheers!!!
Kasiram.
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The following message was posted to: PharmPK
Hi Nageswar,
I have a point to add in addition to what I posted earlier
considering the
additional information you provided in the following message:
>I would not think that the split peak is an impurity.
>Because, I am using MRM method which you all know that highly
specific.
>Moreover, I have injected the standard solution of the compound
>reconstituted in the solvent dried tube and it is also giving the
>split peak.
I still think the contamination on the needle could be the reason as
you are
working on NCEs and it is always possible that your contamination on the
needle could be a closely related NCE which might give similar
transition
pair.
Another possibility is that the contamination on the needle could be
the NCE
that you are actually working with which could be washed into your
sample
during evaporation giving rise to very high concentrations in your
reconstituted sample. When you inject highly concentrated sample
(keeping
all the other chromatographic parameters constant), it is likely that
you
exceed the "capacity factor" of your column which would lead to peak
splitting.
As many others pointed out, degradation of your NCE could be another
likely
possibility.
It seems you got hand full of work to do.
Keep the group posted with your findings.
Good luck,
Kasiram.
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The following message was posted to: PharmPK
Dear group,
Thank you very much for ur thoughtful responses.
I would agree with Mr Kasiram and Dr Manish's suggestions.
I should tell some more things to the group regarding the method.
1) sample volume is 10 uL.
2) sample was reconstituted in the mobile phase.
3) isocratic solvent flow was used for the separation.
4) sample was neither concentrated nor diluted, i.e., reconstituted
in the same volume as plasma (100 uL) from which it is extracted.
5) as Mr Kasiram pointed out correctly, the splitting is not due to
some "general" factors
6)Idid not observe any such peak when the std solution prepared using
the mobile phase injected immediately after the extracted sample
under the similar chromatographic conditions .
7) sample was dried well. no possibility of solvent residues.
First, I would like to try the extraction with different solvents and
try looking in different reconstituting solutions as suggested by Dr
Manish and then, Mr Kasiram's suggestions.
I 'll let the group know with my findings.
Thank you all once again for ur time.
Best Regards,
Nageshwar.
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The following message was posted to: PharmPK
Dear Mr kasiram,
That was a good reasoning. I would like to bring up issue of
"specificity of
MRM" as an additional thread to this discussion.
Most of us do believe MRM is very specific. But I am sure many of us
must
have observed additional peaks at different RT's. If we do not expect an
isomeric analyte to be present in the sample, obviously contribution
from
matrix will be the reason. In that case, MRM detection is not specific.
I have also seen, noisy MRM peaks even at very high S/N peaks. It
will be a
good idea for the forum to through some light on these day today issues.
Thanks,
Vinayak Nadiger
Astra Zeneca India R & D, Bangalore , India
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As to multiple peaks in MRM, consider that sometimes Glu conj.'s (for
example) get busted up by the DP voltage and that a second, faster
eluting peak will be observed as the retention times differ, but not the
apparent masses.
Joyce
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Dear Nagehwar Budha,
Avoid evaporation and try, this may help
Regards,
Martin
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The following message was posted to: PharmPK
Hello everyone,
I have been following up this chain with some interest, if I might
add my
three half pennies to the discussion.
A number of years ago, when in a different job, different company, I was
working on a chemical series of compounds featuring a hydroxamic acid
moiety. When one of these compounds entered tox testing I started noting
split peaks in my MRM chromatograms - however, it was not all
chromatograms
and although it did not occur for standards or QCs (extracted by SPE)
it did
occur for a meaningful number of study samples. As a results of the
randomisation of injection order, it was not obvious that all of the
split
peaks came from 1 female animal at the highest dose administered.
After much
soul searching and testing we concluded that these were real results,
and
the split peaks were indeed likely due to isobaric metabolites of the
test
article (the API, or drug compound that was administered). So we
reduced the
elution strength of the mobile phase and sure enough there were three
discrete peaks. Subsequent fraction collection and NMR analysis
showed them
to be intramolecular re-arrangements of the hydroxamic acid onto the
neighbouring phenyl ring - in two positions. The precursor ion was
the same
and indeed so was the product ion that we were monitoring, hence even
the
selectivity of tandem mass spectrometry did not resolve the
molecules. This
was quite hard to understand at first because it was not male
animals, and
not all the females, and only at the highest doses. It came down to sex
differences and polymorphism in expression of the enzymes performing the
biotransformation (we never did get to the bottom of that).
I would say the thing to take away from this is do not immediately
assume
that you have made mistakes or something is wrong with your
instrumentation.
Instead believe what you are seeing and try to explain it in DMPK
terms. At
the time I thought this example was a bit unusual, but when you look
into
the literature there are other examples.
I would try stretching out your chromatography and seeing what
happens....
good luck!
Phil Clarke
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Would I be correct to postulate that the standard was prepared from
NCE not
incubated with plasma? (i.e. the source of the drug is not some
biological
matrix?) I am theorizing that these are samples from some PK/PD study.
Have you considered oxidation/reduction reactions related to your
solvent of
choice? (or it's impurities?)
Additionally, would I be correct to postulate that the peak was seen
as a
shoulder rather than a new, clearly defined, similarly eluting peak? Do
modified chromatographic parameters more clearly resolve the peaks?
What form of detection are you using? For instance, if using a diode
array
UV visible detector - is the absorbance for both peaks similar over the
entire spectrum?
Is the sample prepared from the same starting material as the
standard? Any
isomerism?
Good luck,
James
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The following message was posted to: PharmPK
Another good point,
Polymorphism on NCE's.
James
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The following message was posted to: PharmPK
When you add the peak areas of your split peaks does this (roughly)
amount to the area you'd get from injecting the standard (single peak)?
Have you tried a different MRM (if that's possible with your compound)?
Do you see the peak splitting when using single stage MS?
Kind regards,
Frederik Pruijn
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The following message was posted to: PharmPK
Are there COOH, sulfate or phosphate moieties in the structure?
Is the molecule low MW and very polar?
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