Kertesz Vilmos, Weiskittel Taylor M, Vavrek Marissa, Freddo Carol, Van Berkel Gary J
Mass Spectrometry and Laser Spectroscopy Group, Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831-6131, USA.
ORISE HERE Intern, University of Tennessee, Knoxville, TN, 37996, USA.
Rapid Commun Mass Spectrom. 2016 Jul 30;30(14):1705-1712. doi: 10.1002/rcm.7607.
Currently, the absolute quantitation aspects of droplet-based surface sampling for tissue analysis using a fully automated autosampler/high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) system have not been fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from tissue sections.
Adjacent tissue sections of propranolol-dosed mouse brain (10-μm-thick), kidney (10-μm-thick) and liver (8-, 10-, 16- and 24-μm-thick) were obtained. The absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and MS/MS analysis. These values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach.
Extraction efficiency of propranolol using 10-μm-thick brain, kidney and liver tissues using droplet-based surface sampling varied between ~45 and 63%. The extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 μm to 24 μm. Selecting half of the samples as standards, the precision and accuracy of propranolol concentrations were determined for the other half of the samples that were employed as a quality control data set. The resulting precision (±15%) and accuracy (±3%) were within acceptable limits.
Quantitation of adjacent mouse tissue sections of different organs and of various thicknesses by droplet-based surface sampling in comparison with bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided satisfactory quantitation accuracy and precision for assay validations. Thus, once the extraction efficiency was calibrated for a given tissue type, tissue thickness and drug, the droplet-based approach provides a non-labour-intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.
目前，使用全自动自动进样器/高效液相色谱/电喷雾电离串联质谱（HPLC/ESI-MS/MS）系统对基于液滴的组织分析表面采样的绝对定量方面尚未得到充分评估。需要了解提取效率及其重现性，以判断该方法从组织切片中绝对定量分析物的潜力。
获取普萘洛尔给药小鼠脑（10μm厚）、肾（10μm厚）和肝（8μm、10μm、16μm和24μm厚）的相邻组织切片。使用标准的大块组织提取方案以及随后的HPLC分离和MS/MS分析，测定连续切片的组织打孔中普萘洛尔的绝对浓度。这些值用于通过基于液滴的表面采样方法确定普萘洛尔从组织中的提取效率。
使用基于液滴的表面采样，10μm厚的脑、肾和肝组织中普萘洛尔的提取效率在约45%至63%之间变化。随着肝厚度从8μm增加到24μm，提取效率从约65%降至约36%。选择一半的样品作为标准品，对用作质量控制数据集的另一半样品测定普萘洛尔浓度的精密度和准确度。所得精密度（±15%）和准确度（±3%）在可接受范围内。
与组织打孔的大块提取相比，通过基于液滴的表面采样对不同器官和不同厚度的相邻小鼠组织切片进行定量分析表明，前一种方法的提取效率不完全，且取决于器官和组织厚度。然而，一旦确定并应用提取效率，基于液滴的方法为分析验证提供了令人满意的定量准确度和精密度。因此，一旦针对给定的组织类型、组织厚度和药物校准了提取效率，基于液滴的方法提供了一种非劳动密集型且高通量的手段，以获取同一类型多个样品的空间分辨定量分析。发表于2016年。本文为美国政府作品，在美国属于公共领域。
