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The following message was posted to: PharmPK
Hello my group back here is working with depot injections and they
have a query about detrmination of drugs in tissues particularly depot
injections.
How extraction of drug has to be done from tissues and how to prepare
the calibration curve in blank tissues to prepare the calibration
curve, using LCMS/MS
Any references or papers will be highly appreciated
Gagandeep Singh & Sukhbir Singh
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The following message was posted to: PharmPK
Dear Sukhbir
Weigh the tissues, prepare the homogenate in water (1:1) and then go
for spiking as for any matrix.
Extract the drug preferbly by precipitation/liquid-liquid method
(sensitivity) and analyse. Prepare a calibration curve.
Similarly samples can be processed and analysed. The volume of
homogenate taken for processing should be constant and final
concentration for each tissue/organ will be calculated for total
homogenate.
Regards
Gurpreet Saini
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The following message was posted to: PharmPK
Dear Gagandeep Singh & Sukhbir Singh
How to extract the drug from the tissue will depend on the
characteristics of your drug. You will always need to homogenize your
sample (turrax) using an appropriate solvent for homogenation. Then,
centrifugate and clean-up your sample, for instance by means of SPE.
Prepare calibration sample by spiking blank tissue.
Regards Bert
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Water is not the best. PBS may be more appropriate. Tissues need to be
thoroughly perfused before processing otherwise the blood content will
bias the result(s).
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
609-228-0243
ed.oconnor.-at-.tandemlabs.com
[Blood in tissue is a problem. Through perfusion can upset
concentration equilibrium, simple blotting is an option, many years
ago we used labeled albumin and corrected for blood concentrations - db]
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The following message was posted to: PharmPK
Dear Mr. Singh,
Extraction and analysis of drugs from tissue samples using LC-MS-MS.
Kindly refer to the following paper published in the journal ANALYST,
2001, 126, 1985-1989. The paper deals with analysis of vet drugs from
animal tissues.
Of course you will find lots of papers if you search for vet drug
analysis and LC-MS-MS.
Hope this helps you.
Regards
Shirish
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I suggest caution when interpreting such studies. Spiking
homogenized tissue to create a calibration curve may not be a true
representation of extraction efficiencies and hence true
concentrations (i.e. extracting drug from perfused tissues is not the
same as 'extracting' drug that has been spiked into a homogenate).
For this reason it is better to (with caution) use such data to
compare levels from different tissues in a semi-qualitative and not
quantitative way.
Best Regards,
Tom
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The following message was posted to: PharmPK
Hi,
I was reading the responses to this topic and noted with interest a
comment made by David Bourne at the bottom of one of the emails.
[Blood in tissue is a problem. Through perfusion can upset
concentration equilibrium, simple blotting is an option, many years
ago we used labeled albumin and corrected for blood concentrations - db]
Are any of these data published? I have often worried about the
issue of blood when we have looked at compound levels in tissues. We
have taken the approach of rinsing organs briefly in PBS and blotting
before snap freezing tissues for later homogenization in PBS. I'm
not sure this is the most appropriate approach, but it is what we
have done. Has anyone tried this approach for cardiac tissue? Or are
there any viable approaches for heart?
Thanks,
Noelle
[It's been awhile. See Triplett, J.W., Hayden, T.L., Mc Whorter,
L.K., Gautam, S.R., Kim, E.E., and Bourne, D.W.A. 1985. Determination
of Gallium Concentration in "Blood Free" Tissues Using a Radiolabeled
Blood Marker. J. Pharm. Sci., 74(9), 1007- 1009 - Once you have blood
mass in tissue mass for your species you can use a drug in blood
concentration to correct for drug in blood in tissue. - db]
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The following message was posted to: PharmPK
>
>Are any of these data published? I have often worried about the
>issue of blood when we have looked at compound levels in tissues. We
>have taken the approach of rinsing organs briefly in PBS and blotting
>before snap freezing tissues for later homogenization in PBS.
We follow a similar approach. In addition to the issues of equilibration
Dr.Bourne suggests, we also think that it is perhaps not possible to rid
the tissue of all the blood given the intricate mesh of cappillaries. We
used radiolabeled inulin to determine the vascular space in brain and
correct for compound due to the presence of blood.
Dai H, Marbach P, Lemaire M, Hayes M, Elmquist WF.
Distribution of STI-571 to the brain is limited by P-glycoprotein-
mediated
efflux. J Pharmacol Exp Ther. 2003 Mar;304(3):1085-92.
Nagdeep Giri
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