Berna M, Murphy A T, Wilken B, Ackermann B
Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA.
Anal Chem. 2002 Mar 1;74(5):1197-201. doi: 10.1021/ac010986a.
The benefits of high-throughput bioanalysis within the pharmaceutical industry are well established. One of the most significant bottlenecks in bioanalysis is transferring in vivo-generated study samples from their collection tubes during sample preparation and extraction. In most cases, the plasma samples must be stored frozen prior to analysis, and the freeze/thaw (F/T) process introduces thrombin clots that are capable of plugging pipets and automated liquid-transfer systems. A new approach to dealing with this problem involves the use of Ansys Captiva 96-well 20-microm polypropylene filter plates to collect, store frozen, and filter plasma samples prior to bioanalysis. The samples are collected from the test subjects, and the corresponding plasma samples are placed directly into the wells of the filter plate. Two Duoseal (patent pending) covers are used to seal the top and bottom of the plate, and the plate is stored at down to -70 degrees C. Prior to sample analysis, the seals are removed and the plate is placed in a 96-well SPE manifold. As the plasma thaws, it passes (by gravity or mild vacuum) through the polypropylene filter into a 96-well collection plate. A multichannel pipet or automated liquid-transfer system is used to transfer sample aliquots without fear of plugging. A significant advantage of this approach is that, unlike other methods, issues related to incomplete pipetting are virtually eliminated. The entire process is rapid since thawing and filtering take place simultaneously, and if a second F/T cycle is required for reanalysis, it is not necessary to refilter the samples (additional clotting was not observed after three F/T cycles). This technique was tested using monkey, rat, and dog plasma and sodium heparin and EDTA anticoagulants. To assess the possibility of nonspecific binding to the polypropylene filter, a variety of drug candidates from diverse drug classes were studied. Validation data generated for two Lilly compounds from distinct classes, before and after filtering, are presented in this paper as practical examples of this technique. While LC/MS/MS is the primary method of bioanalysis in our laboratory, the technique presented in this paper is applicable to other forms of detection as well.
高通量生物分析在制药行业中的优势已得到充分证实。生物分析中最显著的瓶颈之一是在样品制备和提取过程中，将体内生成的研究样品从收集管中转移出来。在大多数情况下，血浆样品在分析前必须冷冻保存，而冻融（F/T）过程会产生能够堵塞移液器和自动液体转移系统的凝血块。一种解决此问题的新方法是使用安赛蜜Captiva 96孔20微米聚丙烯滤板在生物分析前收集、冷冻保存和过滤血浆样品。从受试对象收集样品后，将相应的血浆样品直接放入滤板的孔中。使用两个Duoseal（专利申请中）盖子密封板的顶部和底部，然后将板储存在低至-70摄氏度的环境中。在样品分析前，取下密封盖，将板放入96孔固相萃取歧管中。当血浆解冻时，它会（通过重力或轻度真空）通过聚丙烯滤膜进入96孔收集板。使用多通道移液器或自动液体转移系统转移样品等分试样时无需担心堵塞问题。这种方法的一个显著优点是，与其他方法不同，与移液不完全相关的问题几乎可以消除。整个过程很快，因为解冻和过滤同时进行，如果重新分析需要第二个F/T循环，则无需重新过滤样品（在三个F/T循环后未观察到额外的凝血现象）。该技术使用猴、大鼠和狗的血浆以及肝素钠和乙二胺四乙酸抗凝剂进行了测试。为了评估与聚丙烯滤膜非特异性结合的可能性，研究了来自不同药物类别的多种候选药物。本文给出了来自不同类别的两种礼来化合物在过滤前后生成的验证数据，作为该技术的实际示例。虽然液相色谱/串联质谱是我们实验室生物分析的主要方法，但本文介绍的技术也适用于其他形式的检测。
