Department of Drug Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research, One Health Plaza, East Hanover, NJ 07936, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Feb 15;878(5-6):583-9. doi: 10.1016/j.jchromb.2009.12.031. Epub 2010 Jan 4.
Analyte loss due to non-specific binding, especially container surface adsorption, is not uncommon in the quantitative analysis of urine samples. In developing a sensitive LC-MS/MS method for the determination of a drug candidate, BAF312, in human urine, a simple procedure was outlined for identification, confirmation and prevention of analyte non-specific binding to a container surface and to recover the 'non-specific loss' of an analyte, if no transfer has occurred to the original urine samples. Non-specific binding or container surface adsorption can be quickly identified by using freshly spiked urine calibration standards and pre-pooled QC samples during a LC-MS/MS feasibility run. The resulting low recovery of an analyte in urine samples can be prevented through the use of additives, such as the non-ionic surfactant Tween-80, CHAPS and others, to the container prior to urine sample collection. If the urine samples have not been transferred from the bulk container, the 'non-specific binding' of an analyte to the container surface can be reversed by the addition of a specified amount of CHAPS, Tween-80 or bovine serum albumin, followed by appropriate mixing. Among the above agents, Tween-80 is the most cost-effective. beta-cyclodextrin may be suitable in stabilizing the analyte of interest in urine via pre-treating the matrix with the agent. However, post-addition of beta-cyclodextrin to untreated urine samples does not recover the 'lost' analyte due to non-specific binding or container surface adsorption. In the case of BAF312, a dynamic range of 0.0200-20.0 ng/ml in human urine was validated with an overall accuracy and precision for QC sample results ranging from -3.2 to 5.1% (bias) and 3.9 to 10.2% (CV), respectively. Pre- and post-addition of 0.5% (v/v) Tween-80 to the container provided excellent overall analyte recovery and minimal MS signal suppression when a liquid-liquid extraction in combination with an isocratic LC separation was employed. The compound was stable in 0.5% Tween-80 treated human urine QC samples for at least 24 h at room temperature, after three freeze/thaw cycles with storage at < or =-60 degrees C and for at least 3 months when stored at < or =-60 degrees C. The current work could serve as a simple example in trouble shooting non-specific binding or container surface adsorption in quantitative analysis of urine samples.
分析物因非特异性结合而损失，特别是容器表面吸附，在尿液样本的定量分析中并不罕见。在开发一种用于测定候选药物 BAF312 的灵敏 LC-MS/MS 方法时，概述了一种简单的程序，用于鉴定、确认和防止分析物与容器表面的非特异性结合，并在没有转移到原始尿液样本时回收分析物的“非特异性损失”。通过在 LC-MS/MS 可行性运行期间使用新配制的尿液校准标准品和预混合 QC 样品，可以快速识别非特异性结合或容器表面吸附。通过在收集尿液样本之前向容器中添加非离子表面活性剂吐温-80、CHAPS 等添加剂，可以防止分析物在尿液样本中的回收率低。如果尿液样本未从大容量容器中转移，则可以通过添加指定量的 CHAPS、吐温-80 或牛血清白蛋白来逆转分析物与容器表面的“非特异性结合”，然后进行适当混合。在上述试剂中，吐温-80 的成本效益最高。β-环糊精可以通过用该试剂预处理基质来稳定尿液中的分析物。然而，将β-环糊精添加到未经处理的尿液样本中并不能因非特异性结合或容器表面吸附而回收“丢失”的分析物。在 BAF312 的情况下，用人尿验证了 0.0200-20.0ng/ml 的动态范围，QC 样品结果的总准确度和精密度范围分别为-3.2%至 5.1%（偏差）和 3.9%至 10.2%（CV）。在采用液液萃取结合等度 LC 分离时，向容器中预添加和后添加 0.5%（v/v）吐温-80 可提供出色的整体分析物回收率，并最大限度地减少 MS 信号抑制。在室温下，在 0.5%吐温-80 处理的人尿 QC 样品中至少 24 小时稳定，在-60°C 以下进行三次冻融循环后至少 3 个月稳定，在-60°C 以下至少 3 个月稳定。目前的工作可以作为在尿液样本定量分析中解决非特异性结合或容器表面吸附问题的简单示例。
