Deng Yuzhong, Wu Jing-Tao, Lloyd Thomas L, Chi Cecilia L, Olah Timothy V, Unger Steve E
Discovery MAP Group, Bristol-Myers Squibb Company, Wilmington, DE 19880, USA.
Rapid Commun Mass Spectrom. 2002;16(11):1116-23. doi: 10.1002/rcm.688.
In this work, a high-throughput and high-performance bioanalytical system is described that is capable of extracting and analyzing 1152 plasma samples within 10 hours. A Zymark track robot system interfaced with a Tecan Genesis liquid handler was used for simultaneous solid-phase extraction of four 96-well plates in a fully automated fashion. The extracted plasma samples were injected onto four parallel monolithic columns for separation via a four-injector autosampler. The use of monolithic columns allowed for fast and well-resolved separations at a considerably higher flow rate without generating significant column backpressure. This resulted in a total chromatographic run cycle time of 2 min on each 4.6 x 100 mm column using gradient elution. The effluent from the four columns was directed to a triple quadrupole mass spectrometer equipped with an indexed four-probe electrospray ionization source (Micromass MUX interface). Hence, sample extraction, separation, and detection were all performed in a four-channel parallel format that resulted in an overall throughput of about 30 s per sample from plasma. The performance of this system was evaluated by extracting and by analyzing twelve 96-well plates (1152) of human plasma samples spiked with oxazepam at different concentrations. The relative standard deviation (RSD) of analyte sensitivity (slope of calibration curve) across the four channels and across the 12 plates was 5.2 and 6.8%, respectively. An average extraction recovery of 77.6% with a RSD of 7.7% and an average matrix effect of 0.95 with a RSD of 5.2% were achieved using these generic extraction and separation conditions. The good separation efficiency provided by this system allowed for rapid method development of an assay quantifying the drug candidate and its close structural analog metabolite. The method was cross-validated with a conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay.
在本研究中，描述了一种高通量、高性能的生物分析系统，该系统能够在10小时内提取并分析1152份血浆样本。使用与Tecan Genesis液体处理仪相连的Zymark轨道机器人系统，以全自动方式同时对四个96孔板进行固相萃取。萃取后的血浆样本通过四进样器自动进样器注入四根平行的整体柱进行分离。整体柱的使用使得在相当高的流速下能够实现快速且分离度良好的分离，而不会产生显著的柱背压。这使得在每根4.6×100 mm的柱上使用梯度洗脱时，总色谱运行周期时间为2分钟。来自四根柱的流出物被导向配备有索引四探针电喷雾电离源（Micromass MUX接口）的三重四极杆质谱仪。因此，样品提取、分离和检测均以四通道平行方式进行，从而使血浆样品的整体通量约为每个样品30秒。通过提取并分析十二块96孔板（1152份）不同浓度地西泮加标的人血浆样本，对该系统的性能进行了评估。四个通道以及12块板上分析物灵敏度（校准曲线斜率）的相对标准偏差（RSD）分别为5.2%和6.8%。在这些通用的提取和分离条件下，平均提取回收率为77.6%，RSD为7.7%，平均基质效应为0.95，RSD为5.2%。该系统良好的分离效率使得能够快速开发一种定量候选药物及其结构类似代谢物的分析方法。该方法与传统液相色谱/串联质谱（LC/MS/MS）分析方法进行了交叉验证。
