Xu Yang, Willson Kenneth J, Musson Donald G
Merck Research Laboratories, Clinical PK/PD, West Point, PA 19486, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Feb 15;863(1):64-73. doi: 10.1016/j.jchromb.2007.12.017. Epub 2008 Jan 4.
On-line extraction assays using cohesive high-turbulence liquid chromatography (HTLC) coupled with tandem mass spectrometer (MS/MS) have been developed for the determination of MK-0974 in human plasma and urine. In this report, a four-step strategy for efficient method development of an on-line extraction assay was discussed. Several challenges - namely extraction recovery, carryover and analyte loss to urine container - were addressed. The assay procedures included sample preparation on a Packard MultiPROBE II liquid-handling system, direct injection on a Cohesive Flux 2300 system, on-line extraction with a Cohesive C(18) column (0.5mmx50mm, 50microm), HPLC separation on a FluophasePFP column (50mmx3mm, 5microm) under cohesive quick-elution mode and MS detection on a Sciex API4000 in multiple-reaction monitoring (MRM) mode using positive ionization and turbo ion-spray. Since 37-80% analyte loss was observed in urine QCs, 1% BSA was added to bring urine QC accuracy back to approximately 100% of nominal. Because of the nature of BSA, the urine assay was established by adapting the plasma method. Thus, the two assays were able to be validated side-by-side, which reduced the validation time by approximately two-fold. The linear dynamic ranges were 0.5-500 and 1-1000nM for the plasma and urine assays, respectively. Obtained from five standard curves constructed with five lots of human plasma or urine, the intra-day precision (%CV) was <3.14 and <2.62%, and the accuracy was 98.3-101.0 and 99.13-100.64% of nominal for plasma and urine assays, respectively. Both plasma and urine QC samples were stable when kept at room temperature for 4h, at -70 degrees C for 3 weeks, or after three freeze-thaw cycles. Both assays gave reasonable relative recovery (>88.8%) and acceptable matrix effect (<15%). The carryover from the upper limit of quantification (ULOQ) was able to be controlled at <20% of lower limit of quantification (LLOQ).
已开发出使用具有粘性的高湍流液相色谱（HTLC）与串联质谱仪（MS/MS）联用的在线萃取分析方法，用于测定人血浆和尿液中的MK-0974。在本报告中，讨论了一种用于在线萃取分析高效方法开发的四步策略。解决了几个挑战，即萃取回收率、残留以及分析物向尿液容器的损失。分析程序包括在Packard MultiPROBE II液体处理系统上进行样品制备、在具有粘性的Flux 2300系统上直接进样、使用具有粘性的C(18)柱（0.5mm×50mm，50μm）进行在线萃取、在具有粘性的快速洗脱模式下在FluophasePFP柱（50mm×3mm，5μm）上进行HPLC分离以及在Sciex API4000上以多反应监测（MRM）模式使用正离子化和涡轮离子喷雾进行MS检测。由于在尿液质量控制样品中观察到37%-80%的分析物损失，添加1%的牛血清白蛋白（BSA）以使尿液质量控制的准确度恢复到标称值的约100%。由于BSA的性质，通过调整血浆方法建立了尿液分析方法。因此，两种分析方法能够并行验证，这将验证时间减少了约两倍。血浆和尿液分析的线性动态范围分别为0.5-500和1-1000nM。从用五批人血浆或尿液构建的五条标准曲线获得的数据显示，日内精密度（%CV）血浆分析小于3.14%，尿液分析小于2.62%，准确度血浆分析为标称值的98.3%-101.0%，尿液分析为标称值的99.13%-100.64%。血浆和尿液质量控制样品在室温下保存4小时、在-70℃下保存3周或经过三个冻融循环后均稳定。两种分析方法均具有合理的相对回收率（>88.8%）和可接受的基质效应（<15%）。从定量上限（ULOQ）的残留能够控制在定量下限（LLOQ）的<20%。
