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Dear all,
We are facing blank interference in plasma samples (K2EDTA,
K3 EDTA, Na Heparin). We already tried with different mobile phases
(buffers:acetic acid, formic acid, ammonium acetate and ammonium
formate with pH variation 2.5 to 7 and solvents : methanol and
acetonitrile). with different columns (C18 and cyno) on API4000 LC-MS/
MS instrument.
Sample processing procedures also changed like LLE and SPE (MCX
cartridges) but still problem occuring.
Instead of plasma matrix we tried with milli Q water as a matrix but
still interference observed. Any body will suggest how to overcome
this interfrence from plasma blank.
regards
jaswant reddy
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The following message was posted to: PharmPK
What m/z is the interference that you are seeing? Is it one peak or a
series?
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The following message was posted to: PharmPK
You are also getting it using water as the blank. Not a plasma issue.
1) can you tune it out but still get your analyte response?
2) what its mass compared to the mass of the analyte?
3) are the daughter ions of the interferent and analyte identical?
4) if you wash(condition) the column between injections do you
still get the response
you may have an accumulation on the column that is soluble when
a more polar injection is made-sample (low probability but) see 3
above
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
609-228-0243
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Dear Jaswanth,
I think this problem has nothing to do with chromatography. You can
change all the peek tubings and increase the source exhaust gas. Also
go for the cleaning of source on regular interval basis for this
project. If the daughter ion is less than 100 m/z then if possible go
for some higher m/z.
Hope this will help.
Dr. Mandar Mote
Head Bio-analytical
CRO
Cadila Pharmaceuticals Ltd.
Ahmedabad
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Dear Reddy,
As described, you have tried all possible trials i.e. LLE, SPE
and mobile phase compositions and even matrix replaced with aqueous
showed interferences. In my opinion one probable reason for this
problem can be saturation of your auto sampler with the analyte. I
have faced problems of carryover in some cases when carryover
response was significant even after several blank runs. You can try
with a different auto sampler (If possible) or with changing the
rinsing solution.
Hope this will help you.
Kuldeep Sharma
Scientist
Pliva Research Center
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Dear Jaswanth!.
May be the analyte you are working or the one that you have
previously worked with has some sticky properties. So try to replace
the tubing, change the autosampler. If there is column sticking,
Increase /decrease the acidity the mobile phase and then you can try.
Good idea is to have a needle wash with 50% isopropono and 1%
Triethanol amine. And then proceed
Hope this helps.
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Dear All
I am very much thank full for your suggestions.
Drug(Betahistine) m/z ions 137.1>94.0 and IS m/z ions 151.1>58.1
We got another m/z ion 106 as daughter ion for drug, but that ion
response is not sufficient for our LOQ (10pg).
We tried with following trials as follows...
1.changing peak tubings
2.Cleaninig of source regularely
3.Changing rinsing solution daily
4.Changing with new columns
5.Different washing solutions (which were not effecting recovery) in SPE
6. Fresh auto sampler vials for sample loading
7. Fresh mobile phase every time
Finaliy we concluded that there was no any effect by doing above
trials and also there was no carry over observed for both aqs and
plasma samples.
But when we injecting extracted plasma samples we are facing blank
interference problem, after completing the above (1 to 4) trials also.
please suggest me for better improvement...
regards
jaswanth reddy
Bio anlytical scientist
Apotex research PVT ltd
Banglore
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The following message was posted to: PharmPK
Dear Jaswant Reddy,
To diagnose the problem, inject some of the mobile phase that you are
actually using at the time. This type of blank will indicate if there
is carry-over from some part of the instrument, as mobile phase
injected into mobile phase should not produce peaks. If it does, then
some part of the instrument is introducing carry-over and should be
cleaned or replaced.
Regards,
Frank
Frank Bales, Ph.D.
frankbales.-a-.msn.com
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The following message was posted to: PharmPK
Dear Jaswanth,
I am not sure why you are planning to develop a method for betahistine
but below answers may help you something.
As per the product monograph, betahistine is not easy to measure in
plasma and you have to measure its metabolite 2-pyridyl acetic acid.
But if you still want to develop a method and if it is not a
interference peak may be try to separate the peaks with different mobile
phases.
Hope this will help.
Regards,
Nagesh
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The following message was posted to: PharmPK
1) Can you tune to get a better signal at 106?
2) can you alter the extraction to improve the 106 recovery
3) can you derivatize the betahistine to bias the recovery?
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
609-228-0243
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The following message was posted to: PharmPK
Hi Jaswanth
I am sorry I didnt saw the first mail with the actual problem but was
following the suggestions and from those it appears that you have
some kind of interference from matrix which is correlated with carry
over or contamination. I would like to ask you the following;
1. Did you see the interfering peak by just injecting the blank
extracted sample ( just water extracted with the whole procedure)
prior to the extracted samples- if yes its the contamination( since
you clearly discarded the possibility of carry over by taking all the
precautions) which is coming from somewhere during the processing-
try to explore each solvents or apparatus used in the processing. If
no you are free of any contamination.
2.What is the matirx? Did you tried different lots of the matrix? It
may be related to a specific lot.
3. If your blank extract is free of any peak wether its injected
prior to or after the matrix extracted sample its defenitely coming
from the matrix and in that case I dont want to say but my dear
freind you may have to work on extraction or chromatography to reduce/
eliminate/ seperate the peak from your analyte.
I know its tough to deal with such an issue.
Good luck
Bye
Bajpai
(Lakshmikant Bajpai)
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Dear All,
Thanks for giving your valuble suggestions. We have alredy
developed and validated the method for 2-PAA (metabolite). But our
people want a method for Betahistine.
We received two different sets of ten K2EDTA plasma matrices on
different dates.We performed our experiment on both the plasma lots
(20). One of the plasma lot set(10)was giving lesser interference
than compared to other. Can we proceed to method validation with the
former lots?. I request you suggest what i can do here on.
Thanks & Regards
Jaswanth Reddy
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The following message was posted to: PharmPK
Unless thng have changed dramatically you will need to increase your LOD
based on the background, then select an LLOQ that is appreciably higher
than the LOD. That may get circuitous since your PK fellows may want a
lower LLOQ. And this assumes your intereference is constant. Is it?
Does it increase with increasing concnetrations of betahistine or does
it remain constant?
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
609-228-0243
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