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Dear all,
I have two questions related ti the bioanalysis for Bioequivalence
studies.
1. Which is the proper way to prepare calibration standarts and quality
control samples - gravimetricaly or volumetricaly and are there any
rules recommended?
2. Should the calibration standarts be spiked every day or they could
be prepared before method validation and stored together with QC and
clinical samples in a deep freezer?
Thanks in advance!
Svetlana Stoyanova
Research Associate
Bioequvalence study Lab.
Sofia, Bulgaria
e-mail: SvetStoyanova.-a-.hotmail.com
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Standards can be prepared either gravimetically or volumetrically.
Volumetric methods are more common, as we all know from our student
days. Gravimetric methods are usually reserved for special
circumstances, such as viscous or dense fluids. Regardless, the method
should be validated in accordance with rules of the relevant regulatory
authorities. The important parameter is the recovery.
Calibration standards can be prepared in advance and stored at room
temperature or in a refrigerator, as long as this practice has been
proven in advance by a stability study. Sometimes, because we are all
in a hurry, it is demonstrated that storage for a week, or so, is
acceptable, so that the study can start. Then, during that first week,
the standards are re-analyzed. If they are OK, then that demonstrates
two weeks of stability, etc. In this way, the stability is demonstrated
one week in advance.
Storage in a freezer is more complicated, because the number of
freeze/thaw cycles must be tested and controlled, as well as the rate
of thawing. (Sometimes good, commercial drug products cannot withstand
3 or 4 freeze/thaw cycles.) Freezing is often best done in several
small containers; so that one container is only thawed once, and then
the entire contents used. This practice is must be validated as well.
Frank Bales, Ph.D.
Senior Regulatory Consultant
Worldwide Regulatory Affairs
PAREXEL Intl.
Email: frank.bales.-a-.parexel.com
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The following message was posted to: PharmPK
Dear Colleagues,
I have follow-up questions/comments re preparation of calibration
standards
in support of analysing samples from, for example, a bioequivalence
study.
I have always set up calibration standards on the day of analysis by
spiking
ìblankî plasma with appropriate low volumes of the analyte in a suitable
stock solution. I have assayed QC plasma samples (3 concentrations)
stored
in aliquots under the same conditions as samples, in each assay to
monitor
the performance of the assay. Before commencing routine analysis, I have
also conducted stability assessments to ensure that the analyte is
stable
when stored under the conditions (temperature, time etc.) used for
storing
samples.
However, I can see the advantages of preparing calibration standards in
plasma in bulk and then storing aliquots under identical conditions as
used
for storing samples. This procedure is likely to speed up the analysis
but,
probably more important, can eliminate the possibility of individual
errors
in preparing the calibration standards which could mean that an
analytical
batch has to be re-analysed (if there is sufficient sample remaining),
thus
delaying conclusion of the study.
I have questions about the second approach regarding stability testing
and
the role of QCs. One could argue that if calibration standards are
stored
under identical conditions as used for storing samples there is no need
to
perform a stability assessment. However, I believe that it would be good
practice to include stability testing and I suspect that regulatory
authorities (if relevant) would demand to be able to review such data.
I can
see no alternative to testing stability by analysing plasma samples
containing known concentrations of analyte and stored for various times
at
the conditions used for storing the test samples, using freshly prepared
calibration standards to construct the reference calibration curve. Is
it
reasonable to use a different method of preparing calibration standards
for
the stability testing experiments from the method used for routine
analysis
of test samples?
If calibration curves are constructed using stored calibration
standards, I
have a difficulty in appreciating the role of the QCs. If both
calibration
and QC samples are stored for similar times under the same conditions,
it
would seem to me that the QCs would not monitor assay performance and
would
effectively just provide additional calibration points at slightly
different
concentrations to those used in the calibration curve proper. If, as I
would
hope, the calibration and QC samples were prepared from different stock
solutions of analyte by a different analyst, the analysis of QC samples
would provide a means of double-checking quantitative data but I wonder
if
this is the expected role of QCs. Would the assay-acceptability criteria
requested by the FDA (ìAt least 67% (four out of six) of the QC samples
should be within 15% of their respective nominal (theoretical) values;
33%
of the QC samples (not all replicates at the same concentration) can be
outside the ±15% of the nominal value.î etc.) be relevant for assays in
which the calibration curves are generated using stored calibration
standards?
I should be interested in the consensus view of list members on which
method
of preparing calibration standards is recommended.
Regards,
John Albright
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The following message was posted to: PharmPK
We have discussed this on numerous occasions. While preparing batch
standards would be an efficiency given that there is stability. However
running fresh curves does give a certainty. If QC and Curve materials
are
stored the same way and both decrease or increase, how will you know?
Yes
you can present response changes but treating them separately (Fresh vs
frozen) guards against being blinded.
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The following message was posted to: PharmPK
Dear All,
I had the same problem in the past; we adopted the solution to perform
a stability program for stored samples (cal. and QC)evaluating the
single concentration against a fresh prepared calibration curve and QC.
We enstrablished, case by case, the maximum storage time and the
maximun concentration decrease or increase (including degradation as
well as analytical error of 5% with an evaluated CV for the analytica
performances of about 2-3%)
All the best
Fabio Dr. Macchi
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)