Lásko Zuzana, Hájek Tomáš, Jirásko Robert, Peterka Ondřej, Šimek Petr, Schoenmakers Peter J, Holčapek Michal
Department of Analytical Chemistry, University of Pardubice, Faculty of Chemical Technology, Studentská 573, Pardubice 53210, Czech Republic.
Biology Centre of the Czech Academy of Sciences, České Budějovice 370 05, Czech Republic.
Anal Chem. 2024 Dec 10;96(49):19439-19446. doi: 10.1021/acs.analchem.4c03946. Epub 2024 Nov 27.
Multidimensional chromatography offers enhanced chromatographic resolution and peak capacity, which are crucial for analyzing complex samples. This study presents a novel comprehensive online multidimensional chromatography method for the lipidomic analysis of biological samples, combining lipid class and lipid species separation approaches. The method combines optimized reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) in the first dimension, utilizing a 150 mm long C18 column, with ultrahigh-performance supercritical fluid chromatography (UHPSFC) in the second dimension, using a 10 mm long silica column, both with sub-2 μm particles. A key advantage of employing UHPSFC in the second dimension is its ability to perform ultrafast analysis using gradient elution with a sampling time of 0.55 min. This approach offers a significant increase in the peak capacity. Compared to our routinely used 1D methods, the peak capacity of the 4D system is 10 times higher than RP-UHPLC and 18 times higher than UHPSFC. The entire chromatographic system is coupled with a high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) in both full-scan and tandem mass spectrometry (MS/MS) and with positive- and negative-ion polarities, enabling the detailed characterization of the lipidome. The confident identification of lipid species is achieved through characteristic ions in both polarity modes, information from MS elevated energy (MS) and fast data-dependent analysis scans, and mass accuracy below 5 ppm. This analytical method has been used to characterize the lipidomic profile of the total lipid extract from human plasma, which has led to the identification of 298 lipid species from 16 lipid subclasses.
多维色谱法可提高色谱分辨率和峰容量，这对于分析复杂样品至关重要。本研究提出了一种用于生物样品脂质组学分析的新型综合在线多维色谱方法，该方法结合了脂质类别和脂质种类的分离方法。该方法在第一维结合了优化的反相超高效液相色谱（RP-UHPLC），使用一根150 mm长的C18柱，在第二维结合了超高效超临界流体色谱（UHPSFC），使用一根10 mm长的硅胶柱，两者均采用粒径小于2μm的颗粒。在第二维采用UHPSFC的一个关键优势在于其能够使用梯度洗脱进行超快速分析，进样时间为0.55分钟。这种方法显著提高了峰容量。与我们常规使用的一维方法相比，四维系统的峰容量比RP-UHPLC高10倍，比UHPSFC高18倍。整个色谱系统与一台高分辨率四极杆飞行时间（QTOF）质谱仪联用，在全扫描和串联质谱（MS/MS）中均采用电喷雾电离（ESI），并具有正离子和负离子极性，从而能够对脂质组进行详细表征。通过两种极性模式下的特征离子、MS高能（MS）信息和快速数据依赖分析扫描以及质量精度低于5 ppm来实现脂质种类的可靠鉴定。这种分析方法已用于表征人血浆总脂质提取物的脂质组学谱，从而鉴定出了来自16个脂质亚类的298种脂质。
