I am looking for literature, which shows the influence of adding organic solvent to whole blood in order to perform sample preparation followed by centrifugation. I would need quantitative information of which solvent in which ratio as well as the relation of G number (of the centrifugation) and duration applied to whole blood in order to precipitate all proteins on to separate them. All papers tend to use high G numbers and a quite short time, but what happens if you use a lower G value for a longer time and what's the difference of using methanol or acetonitrile in a ratio of 1:3 or 1:5. Does anyone know a proper reference which investigated this issue systematically? We are looking for small molecules (metabolites) and pharmaceuticals in whole blood and want to get a minimum of residue protein in the solution by using an optimum G number and duration for centrifugation. Currently our capabilities of centrifugation are limited.
Dear Christian,
we recently developed a protein precipitation method for pharmaceutical in plasma, and it was with 1:3 plasma:Acidified Acetonitrile , the centrifuge was 8000 rpm equal to 6500-7000 xG , duration 10 min was enough for precipitating the proteins. with a recovery higher than 93%.
in your case , centrifuging a whole blood would precipitate the red cells first, so you'll have plasma, then the proteins by adding the proper solvent. And about the ratio of the solvent, u have to check the recovery for your analyte to reach the best ratio of solvent.
I think reducing the g force value with longer time wont have much effect as increasing the g force value , because its not a linear equation, it's quadratic.
Dear Armoudjian
Thanks for the answer! Well I am aware of some standard protocols (we also use them). Generally we would add solvent and vortex prior to the separation of the erythrocytes, since we like also to analyze specific drugs in blood which have a significant protein-binding (such as immunosuppressant). However, for me would be nice to have some references showing the relation between g number, time, solvent type and residue protein (or recovery). My impression is everyone ist just using the maximum speed available in order to avoid problems. I would like to know the borderline values for these factors, without testing them myself. I am pretty sure it has been done before, but I have problems finding an article or book which shows a such systematic evaluation.
Can you recommend such an article or book?
Thanks! Thats a nice article. It's a pitty that tehy are looking at plasma only and do not adress the question of g forces. However since they use only about 1200 g for 10 min it is still very useful for me! I should also look at such studies for plasma rather than whole blood.
We developed a study in which we optimized in serum and plasma the same protocol for protein precipitation, I will enclose the link of the paper and you can found our protocol and in this you can found more references.
Article Study of blood collection and sample preparation for analysi...
Nice article! Thanks! Why did you choose to use 20.200 g for the protein precipitation? Was that the maximum of your centrifuge? Did youever test lower values?
I use this because in recent studies in muy group they use this as the best option to eliminate the protein, if you cant use this, you could do more time, the important is be able to eliminate all the proteína, if you need anything more you can tell me
Well, I agree about using the given centrifuge at the maximum anyway. Regarding the equippen I have at the moment, I wonder whats the difference between 1, 2, 10 or 30 minutes at 1300 g or 3000 g. And how this compares with I centrigugation at 20'200 g for 3 or more minutes. Of course I will have to test it under our extraxtion conditions, but it would be nice to have some specific litature, which shows some values I could compare with. Like size of the particles and residual protein (a bit like in the paper you showed me, but depending ion the certifugation parameters).
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