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

• Amicon centrifugation filters suited for isolating small molecules from proteins.
• Validated LC–MS method for rifampicin and thioridazine in macrophage cell lysate.
• Filters unsuited for separating proteins into fractions.
• Filters unsuited for separating small molecules from nanoparticles and proteins.
• Drug–protein binding disruptor is a decisive factor for compatibilities.

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.

Figure optionsDownload as PowerPoint slide