A critical evaluation of Amicon Ultra centrifugal filters for separating proteins, drugs and nanoparticles in biosamples

Elin Johnsen; Ole Kristian Brandtzaeg; Tore Vehus; Hanne Roberg-Larsen; Vanya Bogoeva; Ornela Ademi; Jon Hildahl; Elsa Lundanes; Steven Ray Wilson

doi:10.1016/j.jpba.2015.12.010

A critical evaluation of Amicon Ultra centrifugal filters for separating proteins, drugs and nanoparticles in biosamples

J Pharm Biomed Anal. 2016 Feb 20:120:106-11. doi: 10.1016/j.jpba.2015.12.010. Epub 2015 Dec 12.

Authors

Elin Johnsen¹, Ole Kristian Brandtzaeg¹, Tore Vehus², Hanne Roberg-Larsen¹, Vanya Bogoeva³, Ornela Ademi¹, Jon Hildahl⁴, Elsa Lundanes¹, Steven Ray Wilson⁵

Affiliations

¹ Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway.
² 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.
³ 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.
⁴ Department of Biosciences, University of Oslo, Blindernveien 31, 0371 Oslo, Norway.
⁵ Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway. Electronic address: stevenw@kjemi.uio.no.

PMID: 26719981
DOI: 10.1016/j.jpba.2015.12.010

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.

Keywords: Centrifugal filters; Fractionation; Nanoparticles; Proteins; Rifampicin; Thioridazine.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Centrifugation / methods
Chemical Fractionation / methods
Chromatography, Liquid / methods
Molecular Weight
Nanoparticles / chemistry*
Pharmaceutical Preparations / chemistry*
Proteins / chemistry*
Proteome / chemistry
Solvents / chemistry
Tandem Mass Spectrometry / methods

Substances

Pharmaceutical Preparations
Proteins
Proteome
Solvents