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Dear all,
How to define/determine LLOQ for endogenous compound that is
administered exogenously as a therapeutic.
Regards
venkat bayya
Associate research scientist
Aizant Drug Research solutions
Hyderabad
[An interesting twist on the LOQ question ;-) It now becomes a
question of signifcant difference from baseline. And what would the
baseline be? Do physiological factors play a role, e.g. circadian
rhythm? - db]
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The first thing that comes to mind (if that is still an option) is to
dose with an isotopically labeled version of the compound (stable or
radioactive), followed by specific detection (LC-MS, or radio-
detection). Good luck..
--
Jan Hendrik Beumer
Pharm.D., Ph.D.
Assistant Professor Pharmaceutical Sciences
School of Pharmacy
The Hillman Cancer Center
Research Pavilion, Suite G.27d
5117 Centre Avenue
Pittsburgh, PA 15213-1863
Email: beumerjh.at.upmc.edu
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Hi,
This has always been an issue. Years ago, I used the same strategy to
assess levels of l-tryptophan in plasma using d4-tryptophan and
d3-tryptophan as analyte and internal standard respectively in plasma
based calibration curves. Provided the calibration ranges are selected
appropriately, this strategy worked well to assess the levels of
endogenous l-tryptophan using LC-MS. One problem was the acceptance of
such method by regulatory agencies.
Luis E. Sojo, PhD.
Associate Director Analytical Development
Bio-Analytical Unit
QLT Inc.
email:lsojo.aaa.qltinc.com
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Dear Luis
Regarding how to set an LLOQ for a therapeutically administered
substance when there are endogenous levels of that substance, there
are a few different options, depending on the specific situation.
One option is to administer isotopically labeled drug, as other have
already mentioned.
A second is to screen a number of individual samples of matrix to
determine a maximum background response, and use that to set the
LLOQ. The LLOQ would probably need to be 5 to 10 times the mean or
the max background response.
A third approach would be to use either mock/synthetic matrix (e.g.,
for serum it might be an isotonic buffer containing appropriate levels
of proteins) or a matrix that has been stripped of the analyte (e.g.,
by charcoal or an affinity column) to make standards and quality
controls. Use the analyte-free standards and controls to set an
appropriate LLOQ. You could verify adequate quantification by
measuring a few real samples before and after spiking with a known
amount of analyte. You could also use pooled matrix supplemented as
needed with analyte for a second type of quality control.
My groups have successfully used such approaches. Clearly document
and justify your decisions, and be prepared for questions from curious
reviewers. The 2001 FDA guidance mentions endogenous analytes, but
does not give much help.
-Tom
Thomas L. Tarnowski, Ph.D.
Bioanalytical Development
Elan Pharmaceuticals, Inc.
800 Gateway Boulevard
South San Francisco, CA 94080
thomas.tarnowski.at.elan.com
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Many thanks to Thomas for the excellent summary of the 3 most applied
options in analyzing and setting LLOQ for endogenous compound.
The limitation of using option 1 is dosing with isotope (stable)
labeled compound can be very expensive and may limit the study LLOQ
when dose with combined material to save material cost or to meet
regulatory guidance (for exp: if radio active label is involved). For
option 2, the LLOQ at 5-10 x of the average or the highest normal
endogenous level may be too high for some compounds and is not
practical if the desired drug effect is to inhibits the level of
endogenous compound in the system (for PD biomarker analysis).
Option 3 (synthetic or alter matrices) is the most friendly and cost
effective approach but I heard several well known CROs had 483 for
using the same strategy in their assay. The guidance clearly stated
that the same matrix (un-altered) should be used for the sample
analysis. A synthetic/altered matrix can be used only after it has
been validated to be equivalent to the nature matrix.
Can anyone share the experience he/she had in proving and validating
the equivalence between a nature and altered matrices for an assay?
Many thanks in advance.
Ta Kung Chen, Ph.D.
Director, Analytical Services
La Jolla Pharmaceutical
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Dr. Chen,
The following papers should be of great use to you:
Lee JW, Weiner RS, Sailstad JM. (2005) "Method validation and
measurement of biomarkers in nonclinical and clinical samples in drug
development. A conference report" Pharm. Res. 22: 499-511
Lee JW, Devanarayan V, Barrett YC, Weiner R, Allinson J, Fountain S,
Keller S, Weinryb I, Green M, Duan L, Rogers JA, Millham R, O'Brien
PJ, Sailstad J, Khan M, Ray C, Wagner, JA. (2006) "Fit-for-Purpose
Method Development and Validation for Successful Biomarker
Measurement" Pharm. Res. 23:2, 312-328
Its interesting that we have been quantifying endogenous compounds for
such a long time but sufficient guidance on method validation of these
types of molecules are unavailable.
In using a "surrogate matrix" for validation purposes, one should
prove "parallelism" in the calibration curves of both the surrogate
matrix and unadulterated matrix. For the experiment...
1) Spike known concentrations, preferably a full calibration curve,
in the unadulterated matrix. Evaluate the method in at least 6
different sources of plasma. Of course you will get significant
intercepts. Assess the slope of the calibration curves matrix effects
and they should be statistically similar.
2) Find a suitable surrogate matrix- generally should be close to the
unadulterated matrix. You can try to dilute until the peak of
interest is removed or strip the matrix of the endogenous analyte.
3) Perform the same calibration experiment as the unadulterated matrix.
The slopes of the calibration curves should be statistically similar
between unadulterated and the surrogate matrix (can use a form of
analysis of variance for assessment). The surrogate matrix
calibration should have an intercept statistically similar to zero.
Good luck and hope this helps.
Satjit Brar
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Isn't it advisable to perform dilution linearity along with
parallelism in such cases?
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Isn't it advisable to perform dilution linearity along with
parallelism in such cases?
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There are a few more layers to the onion. Given that it is an
endogenous compound are there sex, age, prandial and/or circadian
changes associated with it? If there are, the 5X adjustment may be
meaniningless overall and may need to be applied to each of
considerations above.
If the action is to increase the endogenous amount to a certain level,
the before and after measurements may be more meaniningful, with each
pretreatment serving as a "control" for each subsequent timepoint.
It is important to validate that the material added to each individual
sample gives the same sensitivity-response/unit mass. This can be
approached as a standard addtion experiment.
--
Ed O'Connor, Ph.D.
Laboratory Director
Matrix BioAnalytical Laboratories
25 Science Park at Yale
New Haven, CT 06511
Web: www.matrixbioanalytical.com
Email: eoconnor.aaa.matrixbioanalytical.com
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