Johnsen Elin, Brandtzaeg Ole Kristian, Vehus Tore, Roberg-Larsen Hanne, Bogoeva Vanya, Ademi Ornela, Hildahl Jon, Lundanes Elsa, Wilson Steven Ray
Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway.
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.
J Pharm Biomed Anal. 2016 Feb 20;120:106-11. doi: 10.1016/j.jpba.2015.12.010. Epub 2015 Dec 12.
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.
对密理博（®）超滤离心过滤器进行了严格评估，用于各种样品制备，即（a）蛋白质组分级分离，（b）在对细胞裂解物中的小分子进行液相色谱-质谱（LC-MS）测量之前进行样品净化，以及（c）分离载药纳米颗粒和释放的药物，以便在生物样品中进行准确的释放分析。（a）将据称具有不同摩尔质量（MM）选择性（10、30、50和100K）的过滤器组合起来，试图对各种复杂性和浓度的样品进行分级分离。然而，产物的MM截留物/滤液谱令人惊讶地相似，并且这些过滤器不适合用于蛋白质组分级分离。（b）在一种经美国食品药品监督管理局（FDA）指南验证的用于测定巨噬细胞裂解物中抗结核药物利福平和硫利达嗪的LC-MS方法中，离心过滤是唯一的净化步骤。为了获得满意的回收率，还需要一个额外的有机溶剂洗涤步骤（破坏药物/蛋白质结合）。（c）离心过滤器非常适合在简单的水溶液中分离药物和纳米颗粒，但对于生物样品则明显不适用，因为常见的药物-蛋白质结合破坏剂会溶解纳米颗粒或与LC-MS仪器不兼容。
