title: A critical evaluation of Amicon Ultra centrifugal filters for separating proteins, drugs and nanoparticles in biosamples.
abstract: Amicon(®) Ultra centrifugal filters were critically evaluated for various sample preparations, namely (a) proteome fractionation, (b) sample cleanup prior to liquid chromatography mass spectrometry (LC-MS) measurement of small molecules in cell lysate, and (c) separating drug-loaded nanoparticles and released drugs for accurate release profiling in biological samples. (a) Filters of supposedly differing molar mass (MM) selectivity (10, 30, 50 and 100K) were combined to attempt fractionation of samples of various complexity and concentration. However, the products had surprisingly similar MM retentate/filtrate profiles, and the filters were unsuited for proteome fractionation. (b) Centrifugal filtration was the only clean-up procedure in a FDA-guideline validated LC-MS method for determining anti-tuberculosis agents rifampicin and thioridazine in macrophage cell lysate. An additional organic solvent washing step (drug/protein-binding disruption) was required for satisfactory recovery. (c) The centrifugation filters are well suited for separating drugs and nanoparticles in simple aqueous solutions, but significantly less so for biological samples, as common drug-protein binding disruptors can dissolve NPs or be incompatible with LC-MS instrumentation.
authors.affiliation: Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway.
authors.forename: Elin
authors.initials: E
authors.lastname: Johnsen
authors.affiliation: Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway.
authors.forename: Ole Kristian
authors.initials: OK
authors.lastname: Brandtzaeg
authors.affiliation: Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway; Department of Engineering Sciences, University of Agder, Jon Lilletunsvei 9, 4891 Grimstad, Norway.
authors.forename: Tore
authors.initials: T
authors.lastname: Vehus
authors.affiliation: Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway.
authors.forename: Hanne
authors.initials: H
authors.lastname: Roberg-Larsen
authors.affiliation: Department of Molecular Biology of the Cell Cycle, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. George Bonchev Str, bl. 21, 1113 Sofia, Bulgaria.
authors.forename: Vanya
authors.initials: V
authors.lastname: Bogoeva
authors.affiliation: Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway.
authors.forename: Ornela
authors.initials: O
authors.lastname: Ademi
authors.affiliation: Department of Biosciences, University of Oslo, Blindernveien 31, 0371 Oslo, Norway.
authors.forename: Jon
authors.initials: J
authors.lastname: Hildahl
authors.affiliation: Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway.
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
authors.affiliation: Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway. Electronic address: stevenw@kjemi.uio.no.
authors.forename: Steven Ray
authors.initials: SR
authors.lastname: Wilson
mesh_terms: D002498:Centrifugation / Q000379:methods; D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D008970:Molecular Weight; D053758:Nanoparticles / Q000737:chemistry*; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D004364:Pharmaceutical Preparations; D011506:Proteins; D020543:Proteome; D012997:Solvents
keywords: Centrifugal filters; Fractionation; Nanoparticles; Proteins; Rifampicin; Thioridazine