Nakai Daisuke, Kumamoto Kazuyo, Sakikawa Chisa, Kosaka Toshiyuki, Tokui Taro
Pharmacokinetics and Drug Delivery Research Laboratories, Sankyo Company, Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan.
J Pharm Sci. 2004 Apr;93(4):847-54. doi: 10.1002/jps.20012.
Ultracentrifugation methods have been widely used for the determination of the free fraction of compounds in plasma, especially for lipophilic compounds. To estimate the effect of contaminated proteins in the "protein-free phase" fraction, 200 microL of human plasma was separated into three layers by ultracentrifugation at 436,000g for 140 min with a table-top ultracentrifuge. Twenty microliters of the middle layer was taken as the protein-free fraction. Major contaminated proteins were analyzed by liquid chromatography/electrospray ionization/tandem mass spectrometry (LC/ESI/MS/MS) and identified as albumin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, apolipoprotein E, and apolipoprotein A-1. alpha-1-acid glycoprotein was not detected. Contamination of albumin was 0.13% of that in plasma. Simulation analysis demonstrated that at an actual free fraction of 1% (protein binding ratio of 99%), the extent of overestimation of free fraction was just 13% and the apparent free fraction was 1.13%. Human plasma protein binding ratios of 10 drugs estimated by this method correlated well with reported values determined by other methods, such as ultrafiltration and equilibrium dialysis, with a correlation factor of 0.98 and a slope of 0.99. Collectively, our results indicate the reliability of this micro-scale ultracentrifugation technique for the evaluation of the protein binding of drugs despite a little contamination of albumin.
超速离心法已被广泛用于测定血浆中化合物的游离分数，尤其是亲脂性化合物。为了评估“无蛋白相”部分中污染蛋白质的影响，使用台式超速离心机在436,000g下超速离心140分钟，将200微升人血浆分离成三层。取中间层的20微升作为无蛋白部分。通过液相色谱/电喷雾电离/串联质谱（LC/ESI/MS/MS）分析主要污染蛋白质，并鉴定为白蛋白、α-1-抗胰蛋白酶、α-2-HS-糖蛋白、载脂蛋白E和载脂蛋白A-1。未检测到α-1-酸性糖蛋白。白蛋白的污染量为血浆中白蛋白量的0.13%。模拟分析表明，在实际游离分数为1%（蛋白结合率为99%）时，游离分数的高估程度仅为13%，表观游离分数为1.13%。用该方法估算的10种药物的人血浆蛋白结合率与超滤和平衡透析等其他方法测定的报道值相关性良好，相关系数为0.98，斜率为0.99。总体而言，我们的结果表明，尽管存在少量白蛋白污染，这种微量超速离心技术在评估药物蛋白结合方面是可靠的。
