Interferences in LC-MS-MS

[Maristela]

Uwe

I thought about deproteinization meaning the precipitation of the proteins. We offten use acetonitrile (100 ul of plasma sample + 200 ul of acetonitrile) better than the temperature.
I read some papers that recommend to precipitate protein using cold acetonitrile instead of room temperature solvent. What the temperature of the solvent could improve the precipitation process ???

Maristela

[Uwe Neue]

Acetonitrile precipitation on its own may help in the protein binding, but you will get a lot of interferences combined with the liquid-liquid extraction. You could consider (just for the experiment to prove the point) a protein precipitation with MeCN (3:1 instead of 2:1) combined with the addition of acid, and evaporation to dryness of the supernatant, reconstitution at pH 11 and extraction. This is complicated, but if it is well executed, the errors can be kept small.

This is with consideration of an experiment that will demonstrate that your losses in your standard technique are due to protein binding. For a valid sample preparation technique for the future, all what you may need to do is to prevent the protein binding.

If you are looking for an alternative technique, my recommndation is SPE.

[Maristela]

Uwe

what acid would add in the precipitation process ? And why evaporate the acetonitrile phase after precipitation instead of transfer to a clean tube, add the amonium and exctract ?

Maristela

[Uwe Neue]

My suggestions still relate to a single (or a few) troubleshooting experiments, not to the situation of the sample preparation technique in general.

The addition of a strong acid before protein precipitation by acetonitrile is meant to disrupt the interaction of the analyte to the plasma protein. You can find background information on this in the literature. Several acids have been used: HCl, H3PO4, H2SO4. We commonly use H3PO4.

You want to evaporate the acetonitrile, since it could affect your extraction results on its own, and the experiment is not clean. It is commonly done under vaccuum, does not take too long, and in the meanwhile, you can do something else.

[Maristela]

Uwe
I will test your suggestions on my next study !
You help me a lot and it was great to talk to you.
As soon as I have some new results I will let you know.
Best regards and thank you very much
Maristela