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what is an internal standard and why it is used in bioanalytical method
development?
--
SK.ABDUL MOHAMMED JAFAR SADIK BASHA
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With the (not unreasonable) assumption that the analyte and IS molecules
are randomly dispersed in liquid matrices at all times, it allows
control over volume changes/losses that occur through say sample
extraction and injection. The idea is to use the IS to track with the
analyte through the assay from start to end. So, the calibrator
standards, seeded controls and unknowns all have the same amount of IS
added. After running these the calibration now becomes a plot of the
peak area or peak height ratios of analyte to IS (y-axis), versus the
concentrations of the calibrator standards (x-axis). The ratio measured
for the unknowns are simply calculated and interpolated from the
calibration plot by whatever process you choose (e.g. visual, weighted
linear regression, etc). One usually chooses an IS to be a close
chemical relative because it is likely to have similar (but not
necessarily identical) solubility/chromatographic/detection behaviour to
the molecule being assayed. It usually should chromatograph at a similar
retention time (before or after) the the analyte but should be totally
resolved from it.
Note, however, that simple assays (e.g. QA work and the like) where
there is very straight forward sample preparation especially with high
precision chromatography injectors and replicate injections (or even
1-step protein precipitaion sample cleanup) may not need an IS at all.
In fact, the use of an IS in those cases may actually increase the
variability compared to that when no IS is used.
Cheers,
BC
Bruce CHARLES
BPharm(Hons), GradDipBusAdmin, PhD, MPS
Associate Professor
Director, The Australian Centre for Paediatric Pharmacokinetics
University of Queensland, School of Pharmacy, QLD 4072
Australia
b.charles.aaa.pharmacy.uq.edu.au
http://www.uq.edu.au/pharmacy/charles.html
http://www.mater.org.au/pharm/accp/index.htm
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Dear Basha,
Internal standard is one which is added in known (and equal) amounts
to all the samples of an analytical run so that any errors in sample
handling can be nullified, as the ratio of analyte to Internal standard
will remain same. For example, you take 200ul of plasma sample
containing some analyte to be determined and you add internal standard
to it before processing the sample for cleanup and extraction which you
may not perform uniformly to all samples. Here comes the internal
standard to your rescue, as the ratio of drug to i.std will be same
even if you make quantitative errors in solvent addition, separation,
reconstitution and sample loading. Also its logical to use an i.std
that will have same physicochemical properties and which preferebly
elutes after the analyte of interest.
I hope this is useful to you.
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I am currently doing some work using hplc to determine the
pharmacokinetic parameters of quinine and chloroquine. I would like
to know whether there is need for an internal standard?. If yes what
is the most appropiate?. Your advice will be highly appreciated
thanks in advance
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Hi there
It is advisable to use an internal standard for the estimation of a drug
from biological sample to reduce the chances of variation in extraction of
drug from biological matrix.. You can definitely try Primaquine as an
internal standard.
Thanks
Ashish Jain
Group Leader - Bioanalytics
Wellquest Clinical Research
Email: rjain.-a-.nicholaspiramal.co.in
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Well Sidney,
It all depends upon the extraction efficiecy and reproducibility achieved and
the number of steps involved in your sample preparation technique. For
compounds with >90% extraction efficiency and single step extraction
procedures, the method can be as well developed without the need for I.S. For
your work, you can decide which of your compounds would need an I.S. and then
go for a compound of that class as an I.S. , which will reflect the
characteristics of your compound. But it is not always necessary for the I.S.
to belong to the same class of compounds. There are reports of Nitrobenzene
being used as an I.S. for Quinine.
It also depends upon the analytical technique, which you are following. If you
are developing a method on LC/MS then you surely need and I.S. For these
purposes isotope labelled compounds are the best internal standards, else,
compounds of the same class can be tried. You got to be real careful here!
You would like to read the following reference:
Simultaneous determination of chloroquine and quinine in human biological
fluids by high-performance liquid chromatography . Jean-FrancoisChaulet et al
Journal of Chromatography: Biomedical Applications
Volume 613, Issue 2, 2 April 1993, Pages 303-310.
You can very well try Sontoquine or Amodiaquine.
All the best.
Rajanikanth M
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Dear Ogwal
I re-echo that there is need to use internal standard
when you are analyzing drugs and metabolites in
biological fluids.
We have successfully used papaverine in the analysis
of chloroquine and metabolites in biological fluids
while we have also used primaquine for the analysis of
quinine and its metabolites in biological fluids.
Please find below the references to both papers:
Ogunbona FA, Onyeji CO, Lawal AA, Chukwuani CM and
Bolaji OO, "Liquid chromatographic analysis of
chloroquine and desethyl-chloroquine in human plasma,
saliva and urine", J Chromatogr, 380:425-430, 1986.
Babalola CP, Bolaji OO, Dixon, PAF and Ogunbona FA,
"Column liquid chromatographic analysis of quinine in
human plasma, urine and saliva" J Chromatogr, 616:
151-154, 1993.
I hope you find the refs useful.
Thank you.
Festus A. Ogunbona
Deprtment of Pharm. Chem.
Faculty of Pharmacy
Obafemi Awolowo University, Ile-Ife
fogunbon.-at-.oauife.edu.ng
or adiogunbona.-a-.yahoo.com
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Dear all,
While doing pk analysis, instead of spiking internal standard to
individual study samples, can we add to the solvent which is used
for the extraction?
Thanks,
B.L.Suresh
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Dear Suresh,
No, we can not do that. The purpose of adding internal
standard is to check variation in the extraction of
the drug of interest. So the internal STANDARD should
also get extracted. If the internal standard is is in
extraction solvent then the sole purpose of adding
internal standard is defeated.
Dr. Sunil bajad
Research Associate
Department of Medical Sciences
School of Vet. Medicine
University of Wisconsin-Madison,
2015 Linden Drive west,
Madison 53706, WI,USA
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Dear Suresh:
I think one of major reasons why internal standard is used is to
compensate the
variation of the extraction procedure. If it is added into the
extraction
solvent prior to the extraction, this function of internal standard
will be
lost.
Yaning Wang
Department of Pharmaceutics
College of Pharmacy
University of Florida
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Dear Suresh,
You can do so provided if you can make sure that you are adding same
volume of IStd spiked solvent to all your samples, which I suppose is a
difficult task. Also the solvents being volatile, may evoporate over
time and your Istd concentration might increase accordingly, which will
lead to erroneous results. Taking all these things into account, it is
better to spike the IStd std directly into the sample matrix.
Regards,
Kasiram.
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Dear Suresh
Adding internal standard(IS) to the extraction solvent may not
partition effectively to the biological fluid containing proteins.
Therefore, adding IS to the extraction solvent for a brief time will
not reflect the exact variation in the extraction process. Many people
add internal standard in organic solvent to the plasma sample. This
process will also affect the extraction process incase if it is in a
higher volume (due to the increase in cosolvent) and the accuracy of
internal standard is compromised if it is in a lesser volume .
The best way to deal this situation is to add the IS in slightly higher
volumes (to be more accurate) in clean glass tubes and evaporate the
organic phase in the tube were you are going to perform extraction
process. To the IS coated tube add the biological fluid and vortex it
strongly so that the IS bind to proteins. Then one can perform
extraction process. Only care to be taken here is the drug should not
bind to the tube material therefore, glass tube would be better.
velpandian
Pharmacokinetics and Metabolism Laboratory
Department of Pharmacology
All India Institute of Medical Sciecnes
New Delhi
India
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The distribution will be different than the analyte but if you have
characterized the difference well.... Ideally the IS would be added at
the
time of collection of the sample. That would allow compensation for
vagaries in handling storage etc as well as processing.... that would
be the
ideal but...
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Dear Suresh
This is in comment to the suggestion made by velpandian. His
suggestion is
true for most cases, however, my work experience with couple of drugs
suggest that there are drugs which have a tendency to bind the walls of
the
glass tube too. so if ur drug is having a tendency to show nonspecific
adsorption to glass surface then the idea of silanizing the internal
surface
of the glass tube could work. In addition not all drugs release form
the
glass surface when dried off their solvent. so make sure that ur drug
releases easily from the glass surface, so that the amount u spike is
completely available to interact with the biomatrix.
Alternatively to avoid inaccuracies in spiking lower volumes (say 20 ?L
or
below), u could also spike 25 or 50 ?L to a larger volume of biomatrix
so
that u do not exceed the spiking volume limit.
hope this helps
All the Best
manish
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)