protein precipitation: organic ratio vs plasma volume



For usual plasma volumes (50-200 ul), the solvent:plasma ratio is often 2:1 or 3:1 (at least with acetonitrile). Now, for lower plasma volumes, for example 5 or 10 ul, Waters recommends that a higher ratio should be used, typically 6:1 or 10:1 (see for instance Ostro SPE FAQ). Do you know why? I wonder if this is related to variable precipitation efficiency or simply for allowing better recovery of the supernatant after centrifugation.

And I wonder another thing: if I would mix 15ul ACN with 5 ul plasma, then add 50 ul water without prior centrifugation, would proteins redissolve or their denaturation would prevent resolubilisation?

Thanks for your comments