I am doing pharmacokinetic/bioavailability study of a drug in plasma samples by HPLC for the first time. Can somebody tell me the sample preparation of plasma for HPLC? Do I have to deproteinized the plasma? In some samples there was hemolysis while collecting, will that interfere?
Hey I suggest you join a short term internship or training in Industry it can clear your many of doubts, I worked with two procedures of plasma drug extraction, liquid-liquid extraction (LLE) and Protein precipitation although solid phase extraction is available and good purity is achieved less prefer due to cost. read principle of LLE and protein precipitation. For LLE use Ethyl acetate as solvent and protein precipitation use Acetonitrile give better result.
Plasma sample specific aliquot and solvent ACN / EA, vortex, centrifuge, take supernatant, do similar with known concentrations (Linearity) and find the unknown concentration plot Y=mX+C, Plot it against time you have a PK profile bell shaped.
Hey I suggest you join a short term internship or training in Industry it can clear your many of doubts, I worked with two procedures of plasma drug extraction, liquid-liquid extraction (LLE) and Protein precipitation although solid phase extraction is available and good purity is achieved less prefer due to cost. read principle of LLE and protein precipitation. For LLE use Ethyl acetate as solvent and protein precipitation use Acetonitrile give better result.
Plasma sample specific aliquot and solvent ACN / EA, vortex, centrifuge, take supernatant, do similar with known concentrations (Linearity) and find the unknown concentration plot Y=mX+C, Plot it against time you have a PK profile bell shaped.
No need to deproteinized plasma if used above procedures, haemolysis will not affect that much you can understand form PK profile.
I hope this article will serve to guide you. Our working group did something similar in serum. Regards
In general, samples that applied to HPLC needs to be of as high purity as possible to avoid blocking HPLC columns and guard columns. Different deproteinization techniques are used.
First, heating denatures most proteins and after heating denatured particles are centrifuged and removed.
Chemical precipitation is more efficient than heating as temperature sensitive compounds can deteriorate in the heating process. Acidification with trichloroacetic acid, centrifugation and neutralization with sodium hydroxide removes 99% of the proteins. Perchloroacetic acid can also be used. Both the acids have some drawbacks. Trichloroacetic acid absorbs strongly below 230 nm whereas perchloroacetic acid leaves salts in solution which can precipitate with organic solvents or contribute to baseline noise of UV detector.
Acetonitrile treatment is also widely used since acetonitrile is a common solvent transparent below 190 nm. Mixing and centrifugation of plasma sample and acetonitrile will lead to precipitation of about 95% of proteins. Non-polar proteins such as albumins remain in liquid phase. The supernatant can be injected directly if a guard column is used to remove the balance 5% of the proteins. On long-term use the guard column will need to be replaced or inverted and washed out to prevent the proteins from entering the main column.
Zaid Alhajjaj good explanation of the concept, but for a beginner like me it is still a bit complicated. I have been using the ACN treatment method for deproteinization of plasma, same volume of plasma and ACN, centrifuging, and then taking the supernatant, diluting with mobile phase then, could we confirm if our plasma is clear now from protein or not?
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology
- Cancer Cells