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Dear Group
I am developing a bioanalytical method for human clinical trials on
LC-MS/MS. The NCE I am working is an acidic molecule with a free COOH
group. I am facing a severe carryover problem (about 50% of LLOQ)
from Injector part of HPLC. I have ruled out all the other parts and
concluded that it is from the injector only. Carryover exists only if
the conc: is about 0.5 ug/mL. I have tried various rinsing solvents
(MeOH , Mobile phase and ACN). but is unable to solve the problem.
The molecule has low solubility and gets precipitated in presence of
water.
I have two questions here.
1. Can any one suggest a solution for this problem or suggest any
other rinsing solvents for such acidic molecule?
2. I appreciate your comments on: "if the carry over is above 50% of
LLOQ and if this happens in more than 50% of placebo treated
subjects, will the data be acceptable to regulatory agencies as the
conc: achieved will all be BLQ" and if so can anyone provide some
references?
Thanks and regards
Vinu
DMPK
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Vinu,
What LC-MS/MS system are you using?
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The following message was posted to: PharmPK
Vinu,
Regarding carryover from injector, try contacting your equipment
vendor. There are often several choices of materials for the
injector, and sometimes for the seal on the injector as well, just
for this reason of reducing carryover.
Regards,
Frank
Frank Bales, Ph.D.
frankbales.-at-.msn.com
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The following message was posted to: PharmPK
Vinu,
Regarding carryover from injector, try contacting your equipment
vendor. There are often several choices of materials for the
injector, and sometimes for the seal on the injector as well, just
for this reason of reducing carryover.
Regards,
Frank
Frank Bales, Ph.D.
frankbales.at.msn.com
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Vinu,
1-What is your injector? Is it a old rotor/stator valve? Try to
locate the
carry-over (valve, needle, needle seat)
I would try an acidic reagent (is your mobile phase acidic?)to
enhance the
solubility of the product in solvent (RCOOH is more soluble in solvent,
RCOO- is more soluble in water).
2-you should/must not have interference higher than 20% of the LLOQ, you
could try to use an higher LLOQ (5 x carryover% x ULOQ)
Hope this may help you.
Fabrice Guillet
Biopredic
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Vinu
Did you tried adding ammonium hydroxide(1-5%) in the mobile phase and
use it as needle wash? If not try it with MP or with IPA/Water
combination.
If it still persists, try doing standard injection prior to each
injection (it will help in washing the injector port and valve prior
to each run.
Hope it will help.
Good luck.
Bajpai
(Lakshmikant Bajpai)
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Dear Vinu,
I've had a similar problem. The compound I was working on was very
lipophilic, basic and poorly water soluble. In larger concentrations,
it was sticking to the column (even with a gradient from 10 to 100%
acetonitrile) and to the peek tubing as well. I replaced the peek
stator of injector valve and the sample loop with a larger stainless
steel one and this seemed to solve the problem with injector
sticking. As for the column sticking - I acidified the mobile phase
and now the carryover is much less pronounced (at least 4 times below
the LOQ).
Also, it might be a good idea to use a needle wash programme, if your
machine has one, or perhaps you can write your own injector
subroutine, where you can order your injector to wash the needle in a
better solvent, for example 50% isopropanol in 1% triethylamine. You
can order your injector valve to stay in the inject position through
the entire gradient run which will hopefully clean the sample loop
efficiently. If you have a large enough sample loop, you can load a
small volume of mobile phase after loading your sample and then load
a small volume of stronger solvent which might also prevent the
compound from sticking to the injector. Be careful, however, when
injecting the stronger solvent at the start of your run: it might
deteriorate your peak shape. If it is very basic, it may shorten your
column life as well.
Hope this was of some value,
best wishes, Jurij Trontelj
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Dear Vinu,
I don't know what are your materials, your mobile phase, and your
washing solvent: Nevertheless, generally, the autosampler carryover
can be reduced by using acidic washing solvent. The best is to use
TFA at 0.05%, but as per their acidic function of your NCE, maybe it
will be not sufficient. A magic washing solvent we currently use is:
acetonitrile/isopropanol/acetone 40/30/30 containing 0.05% TFA. Try
it! It works very well.
Alternatively, you can reduce your dynamic range.
Regards
Bernard
Bernard Jeanbaptiste, MSc
Head of Mass Spectrometry division
SGS Life Science Services, SGS Belgium sa
Vieux Chemin du Poete, 10
B-1301 Wavre (Belgium)
tel ++32 10 421 135
email: bernard.jeanbaptiste.-at-.SGS.com
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Dear Vinu ,
Try mixtures of solvents with DMSO or THF. Replace the rotor seal.
Decrease your ULOQ - you have to dilute your samples more often.
Kind regards
Thomas
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The following message was posted to: PharmPK
Hi,
I have a question here, you suggested to use acidic
solution as washing solvent. Is you compound a base?
Acidic washing solvent works both for acidic and basic
analytes?
Thanks.
Xiaodong Shen
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)