Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Pharmacokinetics, Dynamics and Metabolism, Janssen R&D, Turnhoutseweg 30, 2340, Beerse, Belgium.
Anal Chim Acta. 2018 Aug 22;1020:62-75. doi: 10.1016/j.aca.2018.02.055. Epub 2018 Mar 3.
High-throughput simultaneous quantitative and qualitative (Quan/Qual) analysis is attractive to combine targeted with non-targeted analysis, e.g. in pharmacometabolomics and drug metabolism studies. This study aimed to investigate the possibilities and limitations of high-throughput Quan/Qual analysis by ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS), to develop a widely applicable Quan/Qual UHPLC-HRMS method and to provide recommendations for Quan/Qual method development.
A widely applicable 4.25-min UHPLC method for small-molecules was used to investigate and optimize mass spectrometric parameters of a Synapt G2S for Quan/Qual analysis. The method was applied on a rat metabolomics study investigating the effect of the fasting state and administration of a dosing vehicle on the rat plasma metabolic profile.
Highly important parameters for high-throughput Quan/Qual analysis were the scan mode and scan rate. A negative correlation was found between the amount of qualitative information that a method can provide and its quantitative performance (accuracy, precision, sensitivity, linear dynamic range). The optimal balance was obtained using the MS scan mode with a short scan time of 30 ms. This 4.25-min Quan/Qual analysis method enabled quantification with accuracy and precision values ≤ 20% at the lowest quality control (QC) level and ≤15% at higher QC levels for 16 out of 19 tested analytes. It provided both parent m/z values and fragmentation spectra for compound identification with limited loss of chromatographic resolution and it revealed biologically relevant metabolites in its application to the metabolomics study.
Quan/Qual method development requires balancing between the amount of qualitative data, the quality of the quantitative data and the analysis time. Recommendations are provided for MS resolution, scan mode, scan rate, smoothing and peak integration in Quan/Qual method development and analysis.
高通量同时进行定量和定性（quan/qual）分析具有将靶向分析与非靶向分析相结合的吸引力，例如在药物代谢组学和药物代谢研究中。本研究旨在通过超高效液相色谱（UHPLC）与高分辨率质谱（HRMS）相结合，探索高通量quan/qual 分析的可能性和局限性，开发一种广泛适用的 quan/qual UHPLC-HRMS 方法，并为 quan/qual 方法开发提供建议。
使用一种广泛适用的小分子 4.25 分钟 UHPLC 方法来研究和优化 Synapt G2S 的质谱参数，以进行 quan/qual 分析。该方法应用于一项大鼠代谢组学研究，旨在研究禁食状态和给药载体对大鼠血浆代谢谱的影响。
高通量 quan/qual 分析的重要参数是扫描模式和扫描速率。方法提供的定性信息量与定量性能（准确性、精密度、灵敏度、线性动态范围）之间呈负相关。使用 MS 扫描模式和 30ms 的短扫描时间获得最佳平衡。这种 4.25 分钟 quan/qual 分析方法能够对 19 种测试分析物中的 16 种在最低质控（QC）水平下实现≤20%的准确度和精密度值，在较高 QC 水平下实现≤15%的准确度和精密度值。它为化合物鉴定提供了母离子 m/z 值和碎片谱，在其应用于代谢组学研究时，几乎没有损失色谱分辨率，并且揭示了具有生物学意义的代谢物。
quan/qual 方法开发需要在定性数据量、定量数据质量和分析时间之间进行平衡。为 MS 分辨率、扫描模式、扫描速率、平滑和峰积分提供了 quan/qual 方法开发和分析的建议。
