3 layers after deproteinizing plasma
So I am trying to deproteinize plasma with acetonitrile, but after they spun, it resulted in 3 layers: a bottom precipitate, a middle layer slightly red liquid, and a top clear layer liquid. I understand that the middle layer is most likely red due to lysed red blood cells, but is the top layer just straight acetonitrile? Is the middle layer just blood? Where is the plasma located? My analyte is hydrogen peroxide. Last time I did this (granted, with a different batch of whole blood samples), it turned out exactly as expected with a defined precipitate and one homogenous supernatant. I am trying to stick with organic precipitation options and avoid acid base. I can’t really find anything online about this besides looking up general densities of everything - which ACN is much less dense than plasma, but i was under the impression that the plasma is in solution with the ACN. So is the top layer what I need? Should i just collect both layers either way and vortex before use? Because at this point i do also need to consider the dilution factor. Feel like I’m stress rambling now, any help or guidance would be appreciated.