Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry.

Four categories of protein precipitation techniques (organic solvent, acid, salt and metal ion) were tested in plasma using spectrophotometry to assess protein removal efficiency across a range of volumes, species and lots. Acetonitrile, trichloroacetic acid (TCA) and zinc sulfate were found to be optimal at removing protein in their categories (>96, 92 and 91% protein precipitation efficiency at a 2:1 ratio of precipitant to plasma, respectively). A post-column infusion LC-MS/MS system was used to assess ionization effect of a protein-bound drug caused by the endogenous components remaining after using various protein precipitants. The extent of ionization effect varied with mobile phase (-20 to 93%), protein precipitant (0.3-86%), but only slightly with species (86-93%). The optimal bioanalytical methodologies for removal of plasma proteins and minimal ionization effect for the probe molecule in positive ion turboionspray LC-MS/MS involve the use of TCA for precipitation with mobile phases consisting of either pure organic solvents (methanol:water or acetonitrile:water) or precipitation with all of the mass spectrometer compatible precipitants evaluated with a methanol:aqueous 0.1% formic acid mobile phase.