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Can you please suggest differert procedures for performing stability of analyte in blood after collection for bioanalysis?
Also please elaborate whether, the resulting plasma will give the less response of analyte ?
Thanksand Regards
Shital P.
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Collect whole blood with anticoagulant. Measure Hematocrit. Spike to contain analyte at target concentration in the plasma portion as determined by hematocrit. RUn in parallel to spiked plasma. Run stability in each. Then harvest and compare analyte conc. Recovery may be less in whole blood due to binding to surface receptors, uptake by platelets or cells, or plasma sill associated with red cells. You may need to run a wash of the red cells to recover the plasma associated with the red cells. Wash gently with appropriate solution to avoid hemolysis. If the difference is large you may want to hemolyze another set of samples.
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Dear Shital,
It is better to perform the stability of the assay as a part of the method development. You can spike the blank plasma or serum with different amount of analyte and store under different temperature conditins and analysed periodically.
After sampling you can study the drug concentration in fresh samples collected and after few days of storage at -80C. If drug is not stable then definitely, the response will decrease with passage of time.
Dr Zafar
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The following message was posted to: PharmPK
Dear Shital,
It's a tough job to do stability exp. in blood, as you can't not store blood in deep freezer. Hence it is recommended to complete the whole blood analysis ASAP, but you can carryout BT exp. It is better to analyze whole blood as a blood spot analysis technique. It's not necessary that haemolyzed plasma always gives less response; it depends upon your method.
Regards,
Sudipta Basu
Senior Research Scientist-DMPK
Sai Advantium Pharma, Pune, India
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Hi Shital,
You can improve the stability of analyte in blood by adding some stabilizer (Eg: dichlorvos). Stabilizing agents can improve your analyte stability in blood/plasma. Collect the blood samples into the vials containing anticoagulant prepared in stabilizing agent.
I hope this will help you.
Regards,
Amar
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Dichlorvos is not a universal stabilizer, but particularly for Ester
drugs. Stabilizing agents need to be chosen based on compound chemistry.
Vinayak
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The following message was posted to: PharmPK
Dear Amar,
Dichlorvos is not a universal stabilizer, but it's a pesticide. Hence be careful while using that stabilizer. You can use Dichlorvos, particularly for ester drugs but for amide containing drugs it will not work. You can try other stabilizers like NaF, Bistatin etc. Stabilizing agent needs to be chosen based on compound chemistry.
All these will work very well in plasma but for whole blood exp. needs to be carrying out. Some stabilizers are having partitioning effect.
Regards,
S.Basu
Senior Research Scientist (DMPK)
Sai Advantium Pharma Ltd., Pune, India
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Hi amar,
but the use of stabilizers may affect the drug recovery or in such cases we need to perform Stabiliser's effect
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The easiest way to assess blood stability is to spike analyte to fresh whole blood, to incubate different times at different temperatures (eg 37 degrees, room temp and on ice up to eg 2 hours), then to take aliquots and process them either immediately (protein prec with acetonitrile) or to flash freeze and store in freezer for analysis on another day. Of course, a reference needs to be included that is frozen or extracted at T=0 hours. This will provide data on blood collection stability, assuming that your final assay is in plasma. If you have a whole blood assay, of course you would need to include F/T and Long term stability too.
Another way to do it is same proces, but spin down blood aliquots from stability test to prepare plasma. This is more difficult to do as then blood/plasma distribution comes in the picture too. Concentrations in plasma can be between 0 (i.e. complete distribution into RBC) and 2x (complete distribution into plasma) the spiked concentration in whole blood.
Best Regards, Ronald
J&J Belgium
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To avoid clotting, especially at 37C, an anticoagulant should be selected as well.
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