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Hi all,
I am trying to develop an LC/MS/MS method and during the tuning I saw
my molecular ion signal at (m+H)+ for m/z 292 which is correct and
major fragments at m/z 275 and 159. Here is the issue:
1. Since the molecular ion signal is weak: Could I choose this MRM
pair 275/159? or am I better off choosing 292/159 pair? With
292/159 pair my LOQ could be 10 pg/mL vs with 275/159 pair my LOQ is
5 pg/mL.
Is this acceptable for quantitation purpose?
This method would be used to quantitate human clinical trial
samples. Any CRO input would be greatly appreciated.
Thank a lot.
Sue
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The following message was posted to: PharmPK
Dear Sue,
Do I understand correct: you would like to fragment your analyte in the
source, to obtain fragment 275?
Regards Bert
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hi,
I think your compound molecular weight is 275 most probably.
because if you are using water as solvent Na will interfere so you
have to add molecular weight of Na.
most probably Na is interfering.
regards.
nagarajan.
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The following message was posted to: PharmPK
Dear Sue,
You shouldn't consider 275/159 pair for MRM transition and you should
consider either 292/275 or 292/159,
The second pair (292/159) you should consider only incase if 159 is
coming only from 292.
To achieve the sensitivity you have to optimize all MS, chromatographic
as well as extraction parameters.
Hope this will help,
Regards,
Nagesh
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The following message was posted to: PharmPK
Dear Sue,
I do not think you can do that. This is because the principle use of MRM
mode in LC/MS/MS is to achieve greater selectivity which is not possible
with simple LC/MS. This is because, you have 3 quadrupoles (Q1, Q2 and
Q3) in sequence in LC/MS/MS which lets you select the ion of
interest(formed at the source) using Q1 and fragment it in Q2 and select
a suitable/stable fragment using Q3 which gets detected eventually. So,
by selecting an MRM transition pair (m/z 292/159), you are making the
instrument to quantify the Q3 m/z mass (159 in your case) emanated from
Q1 mass (m/z 292) formed at the source. But, when you select the other
pair (m/z 275/159) you are loosing the selectivity as you are allowing
an m/z ion through Q1 (which is completely different from the parent).
This can be any junk material from your sample matrix. Alternatively, it
is possible that the parent compound may get fragmented at the source
itself producing the secondary ion (m/z 275 in your case). Looking at
the sensitivity you are achieving using the m/z 275/159 pair, it could
be the later that is happening in your case. I think the only condition
under which you can use the m/z 275/159 pair, if at all, is when you
prove that you have no impurity/metabolite matching that mass in your
sample which, I think, is impossible considering the variation in the
composition of samples that you obtain from a large population.
I request experts in this filed to correct me if I am wrong.
Thanks,
Kasiram.
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hi,
I think your compound molecular weight is 275 most probably.
because if you are using water as solvent Na will interfere so you
have to add molecular weight of Na.
most probably Na is interfering.
regards.
nagarajan.
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The following message was posted to: PharmPK
Dear Sue,
I would be wary of using the 275/159 pair since you are relying on
stable fragmentation of 292>275 in the ion source. Given that you will
be looking at (I guess) plasma and urine samples, there would be matrix
effects that could cause variability. I would see if there is any way of
improving the [M+H]+ ion signal first, such as mobile phase buffer,
organic solvent (methanol is usually better than acetonitrile),
declustering/entrance potentials etc. and if that fails you could try
changing the extraction method to go from 10 to 5 pg/mL and stick with
the 292/159 MRM.
I hope that helps.
David
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Hello all,
Thanks very much for your reply. The parent MH+ is at m/z 292. This
is a synthesized material so the molecular ion is at m/z 292, which
is correct.
I agree with Nagesh and Kasiram but I have also heard that not all
but some CRO uses this to run human clinical samples. My question is
what about if there is no intereference at that retention time from
the sample for MRM transition pair (275/159). How to prove it that
there is no intereference from matrix components, impurity or
metabolites? Has anyone developed methtod this way?
Thanks very much
Sue
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The following message was posted to: PharmPK
Dear Sue,
I think you can not go with 275/159 transition pair.
When you want to quantify 159 m/z fragment you have to
make sure that it is generated from your(M+H)292 m/z.
As Kasiram and nagesh said correctly 275 may be
generated from other masses at Q1,which may not be
consistent and reproducible.
More over selectivity would be better if you choose
your M+1 and its most stable fragment.
Hope this may help you,
Regards
Rajareddy
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The following message was posted to: PharmPK
Dear Sue,
Further to what I posted earlier, the other reason for not to opt
fragmenting at source is that you cannot differentiate between parent
and metabolites (especially if both elute at the same time), as
metabolites (if not have similar m/z) may form m/z 275 fragment.
However, you can get away with this by separating parent from
metabolites by adjusting LC conditions which may mean long run times. I
think it is not worth opting 275/159 over 292/159 for the marginal gain
in sensitivity.
Kasiram.
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The following message was posted to: PharmPK
Sue,
It would be useful to know what conditions you are using e.g. mobile
phase composition. Also, just out of interest, what mass spec are you
using?
Regards,
Derek
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Hello,
The problem is that one of my CRO developed, validated method and ran
samples from human clinical trials (about 3000 plasma samples) using
275/159 pair.
I don't know how good is this data. We see accumulation of drug upon
daily dosing and longer half life. I don't know whether it's due to
the method or that's how the compound is behaving in human.
Thanking you, Sue
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The following message was posted to: PharmPK
Sue,
I think the CRO should do some more work - test scans with a few time
points of study samples but a longer run time to show that they separate
the compound from any metabolites that may have the 275 MH+ mass. This
way you can rule out analytical problems by doing some more LC/MS work.
If there is analytical interference from metabolites than you have to
rerun the samples but maybe that is not the case.
Regards,
Laura
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The following message was posted to: PharmPK
Dear Sue,
Please consider the following;
1) The possibility of interference by matrix should be tested in the
development or in the validation of the method. One way is to spike
matrix
from different sources and compare the signal. An other interference is
ion-suppression, which can be tested for. I think you can check the
validation report or contact the CRO.
2) If you suspect a (potential) metabolite coursing interference, it's a
more difficult situation. If the metabolite is described and
synthesized a
test is possible.
Kind regards,
Ben de Rooij
ben.de.rooij.-a-.zonnet.nl
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The following message was posted to: PharmPK
Dear all,
Interfering metabolites could occur at both m/z=275 and 292 (e.g. in
Sue's case) and both masses need to be checked for this possibility.
In addition, it is also possible that both the parent and a metabolite
at m/z=292 give the same main fragment (e.g. m/z=159) and that they run
very close together.
Life, even tandem MS life, is not always that simple.
Kind regards,
Frederik Pruijn
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