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I have a Quattro Micro which is being used to determine basic drugs in
plasma in electrospray positive ion mode by reverse phase HPLC.
Some compounds are linear from 0.5 to 2000 ng/ml (5 ul injected
on-column), but others from 0.5 ng/ml to 1000.
Is there anyway to increase the linearity such as varying the
electorspray voltage, nebulizing gas temperature (desolvation gas),
nebulizing gas temperature, water added post-column (analytes and
metabolites elute at fairly high methanol content), etc??
Thanks for the help.
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I dont know whether you have tried this. Since your injection volume
is 5
ul, I presume you are using a 2.1 mm ID column. I have injected upto
100 ul
sample on this column, depending on the concentration of sample. Some
cases,
peak splitting occurs once we cross 25 ul, irrespective of sample
concentration. If you have sufficient sample, give a try for higher
volume
injection.
(Post column addition of aqueous will help in improving spray
characters at
late stage of gradient, there by increasing sensitivity. But I have
not seen
any quantitative information as to how much it will enhance
sensitivity.)
Thanks,
Vinayak Nadiger
Astrazeneca R & D , Bangalore , India
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Thanks for the reply.
Very good recovery on all my samples.
Calibration curve flattening out at higher concentrations due to there
being more material present than can ionize.
I can dilute samples at higher end, but was hoping to understand an
instrumental parameter I could adjust to increase the ion efficiency at
higher concentrations without loosing sensitivity at lower
concentrations.
James Little
Tel. No. 423-229-8685 office
Tel. No. 423-229-8022 lab
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Hi James
If it is only ionization issue i will suggest you to
try (apart from temp, potentials and gases) changing
the amount of mobile phase you are pumping into the
source or changing the solvent(s) in mobile phase.
hope this may help
nageshwar
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Thanks, I will give that a try.
I want to do a designed experiment of some type and trying to decide
which variables to examine.
James Little
Tel. No. 423-229-8685 office
Tel. No. 423-229-8022 lab
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Hi James
Nageshwar is right, you can try that one.
You can also try to increase the acid or base component of the mobile
phase ( acid for positive ion and base for negative ion) which will
be helpful to increase the ionization. Basically you need to produce
more ions which could be done by increasing the acid or base and will
be further facilitated by the appropriate mobile phase. For example
methanol is much more helpful for a better ionization with APCI than
acetonitrile, though its not always a thumb rule.
Further I hope that you must have checked that your detector is not
getting saturated at higher concn. in which case none of the
suggestions is going to work out.
Hope it will work.
Bye
Bajpai
(Lakshmikant Bajpai)
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The non-linearity (at higher concentrations) with ESI-LCMS is well known
and intrinsic to the technique. Chris G. Enke has written papers that
may be of help to you in this respect. I suggest you have a look at his
website:
http://chemistry.unm.edu/faculty/enke.cfm
Kind regards,
Frederik Pruijn
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Thanks for all the ideas.
No problem with the detector in this range, must be related to the
number of ions that can be formed from the droplets under the conditions
employed.
James Little
Tel. No. 423-229-8685 office
Tel. No. 423-229-8022 lab
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Nice reference, will be helpful in understanding the limitation in my
method.
Might not be able to fix, just wanted to increase linearity another 1/2
order of magnitude if possible.
James Little
Tel. No. 423-229-8685 office
Tel. No. 423-229-8022 lab
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Hi slittle
I am just curious, why do you need to higher than 1000 or in other
cases 2000ng/ml? Your existing dynamic range of 0.5 to 2000 ng/ml
should work for most small molecules. The attempt would be to go
lower rather than going higher. At higher concentrations there are
chances that nonlinearity becomes prominent as is the case described
by you . Linearity is sometimes related to the nature of the drug
molecule itself and/or the process of sample cleanup. Some molecules
do create a lot of trouble with linearity issues at higher
concentrations but there are other ways dealing with it. One can
perform dilution of matrix (higer conc) and support with dilution
QC's to prove that the higher samples are getting diluted linearly
into your calibration range.
Hope this helps
Manish Issar
Sandoz (Formerly Eon Labs Inc.)
4700 eon drive
Wilson, NC-27614
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James Little,
One reason for nonlinearity when using MRM transition
is the formation of the dimers rather than saturation
of your detector (lot of lectures and papers have
mentioned this). If using eletrospray, you are very
lucky to have a danymic range of 0.5-2000ng/mL, and a
danymic range of 500 or 1000 magnitude is typical when
using electrospray.
Therefore, may be the best way is just to dilute your
samples at high concentrations. It is a rule of thumb,
the ULQ' signal should not have a signal with more
than 2 million counts.
If you want to do a decent job, I would not go to
quadraic regrassion.
Those are just suggestions.
XIaodong
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)