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.
Polson
Cara
C
Lilly Laboratory for Bioanalytical Research, 3650 Danforth Avenue, Scarborough, Ont, Canada M1N 2E8.
Sarkar
Pratibha
P
Incledon
Bev
B
Raguvaran
Vanaja
V
Grant
Russell
R
D000818:Animals; D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004285:Dogs; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D051381:Rats; D012015:Reference Standards; D012709:Serum Albumin / Q000032:analysis; D012997:Solvents; D013045:Species Specificity
D001798:Blood Proteins; D012709:Serum Albumin; D012997:Solvents