Faculty of Chemical Technology, Department of Analytical Chemistry, University of Pardubice, Studentská 573, 532 10, Pardubice, Czech Republic.
Anal Bioanal Chem. 2020 Apr;412(10):2375-2388. doi: 10.1007/s00216-020-02473-3. Epub 2020 Feb 20.
Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5 min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.
超高效超临界流体色谱-质谱联用（UHPSFC/MS）在高通量生物样本脂质组定量分析方面具有巨大潜力；因此，本文比较了 UHPSFC/MS 与超高效液相色谱-质谱联用（UHPLC/MS）在亲水作用色谱（HILIC）模式下作为脂质分离的第二种强大技术的全优化和方法验证。首先，考察了六种常见提取方案的性能，其中 Folch 法在回收率、基质效应和精密度方面效果最佳。然后，对 8 种脂质类别的全优化和分析验证采用 UHPSFC/MS 和 HILIC-UHPLC/MS 方法，对相同的样本集进行分析，并应用于人血浆、人血清和 NIST SRM 1950 人血浆混合样本的脂质组学特征分析。选择适当的内标（IS）对每个脂质类别的定量工作流程具有关键重要性，如验证时的选择性和使用每个脂质类别的多个内标计算定量误差。验证结果证实了两种方法的适用性，但 UHPSFC/MS 提供了一些明显的优势，例如成功分离非极性和极性脂质类，而不像 HILIC-UHPLC/MS，总运行时间更短（8 分钟与 10.5 分钟），并且稍微更稳健。各种类型的方法（UHPSFC/MS 和 HILIC-UHPLC/MS）、生物材料（血浆和血清）、IS（实验室和商业混合物）和标准参考物质的文献数据之间的相关性表明，从 8 种脂质类别的定量分析、血清和血浆中的浓度差异以及非商业可得的内标混合物在脂质定量中的适用性方面进行了实验室内部和实验室之间的比较。
