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This is about the calculation of precision in Bioanalytical sample
analysis by HPLC. Precision calculation requires atleast 3-4 injections
of the same concentration from the same vial.
However, if the volume of reconstituted plasma sample is not enough to
inject 3-4 times, how would we go about calculating the precision.
Sandeep.
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Sandeep,
Regarding your question related to the calculation of precision:
It is common to need larger volumes of samples during validation than
is normally available during analysis.
Precision is easy to demonstrate using pooled samples: take several
reconstituted plasma samples and combine them together (well). The
mixture can then be used repeatedly.
Accuracy is usually more difficult to demonstrate, and may require
fortified (spiked) plasma matrix. If this is your case, then Precision
and Range can be determined in the same fashion. As the FDA "Guidance
for Industry, Bioanalytical Method Validation" states, "The accuracy
and precision with which known concentrations of analyte in biological
matrix can be determined should be demonstrated. This can be
accomplished by analysis of replicate sets of analyte samples of known
concentrations_ QC samples _from an equivalent biological matrix. At a
minimum, three concentrations representing the entire range of the
standard curve should be studied: one within 3x the lower limit of
quantification (LLOQ) (low QC sample), one near the center (middle QC),
and one near the upper boundary of the standard curve (high QC)."
Frank
Frank Bales, Ph.D.
Senior Regulatory Consultant
Worldwide Regulatory Affairs
PAREXEL Intl.
2520 Meridian Pkwy, Suite 200
Durham, NC 27713
Email:frankbales.at.msn.com
frank.bales.aaa.parexel.com
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The following message was posted to: PharmPK
Dear Sandeep,
To my opinion "re-injection variability" only contributes to some
extent to
the overall assay variability. Random errors due to spiking reference
and
internal standards, sample-to-sample differences in extraction losses,
differences in matrix effects,...
Therefore, according to the FDA Guidance on Bioanalytical Method
Validation
dd. May 2001, plasma samples are to be spiked at LLOQ, low, medium and
high
concentrations and fully analyzed in (at least) six fold. % CV should be
within 15% (20% at LLOQ).
Kind regards,
Bart Remmerie
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The following message was posted to: PharmPK
You are thinking only of the special case of assessing the (im)precision
of injection volume and the chromatography post-workup. If you want to
look at the (more meaningful) imprecision of the assay at a particular
concentration then the WHOLE assay has to be replicated (> 5 times
preferably).
BC
Bruce CHARLES, PhD
Associate Professor and Director,
Australian Centre for Paediatric Pharmacokinetics
School of Pharmacy
The University of Queensland, 4072 Australia
[University Provider Number: 00025B]
TEL: +61 7 336 53194
FAX: +61 7 336 51688
B.Charles.-at-.pharmacy.uq.edu.au
http://www.uq.edu.au/pharmacy/brucecharles/charles.html
http://www.mater.org.au/pharm/accp.htm
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)