Madörin M, van Hoogevest P, Hilfiker R, Langwost B, Kresbach G M, Ehrat M, Leuenberger H
Novartis Pharma AG, Basel, Switzerland.
Pharm Res. 1997 Dec;14(12):1706-12. doi: 10.1023/a:1012171511285.
The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption.
Field-Flow Fractionation is defined as a group of pseudochromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy.
Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), alpha 2-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothiocyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA.
If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug/plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation time.
研究不对称流场流分级法（Asymmetrical Flow FFF）作为一种替代工具，用于检测亲脂性药物（N-苯甲酰-星形孢菌素）在人血浆蛋白组分中的分布情况，考察其高分离速度以及表面吸附导致的材料损失。
场流分级法被定义为一组准色谱分离方法，其中化合物在外部施加力的影响下，根据其物理化学性质的差异进行分离。该方法用于分离人血浆中的蛋白组分。通过荧光光谱监测分级洗脱液中的药物含量，研究各组分中的药物分布。
在不到十分钟的时间内，人血浆被分离为人血清白蛋白（HSA）、高密度脂蛋白（HDL）、α2-巨球蛋白和低密度脂蛋白（LDL）组分。使用市售蛋白质参考标准品对系统进行校准并鉴定各个组分。发现膜类型和载体溶液组成对N-苯甲酰-星形孢菌素和异硫氰酸荧光素-白蛋白（FITC-白蛋白）的绝对回收率有相当显著的影响。在研究过程中对这两个因素进行了优化。发现N-苯甲酰-星形孢菌素在含有HSA的组分中富集。
如果能精心选择并控制实验条件以抑制分离膜上的药物和血浆蛋白吸附，不对称流场流分级法显示出高回收率且能快速分离人血浆蛋白，并且可以成为表征药物/血浆蛋白相互作用的可靠工具。就分析用途而言，它在易用性、精度和分离时间方面有潜力与超速离心等成熟技术相媲美。
