Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
J Chromatogr A. 2020 Jan 25;1611:460575. doi: 10.1016/j.chroma.2019.460575. Epub 2019 Sep 26.
Improvements in sample preparation, separation, and mass spectrometry continue to expand the coverage in LC-MS based lipidomics. While longer columns packed with smaller particles in theory give higher separation performance compared to shorter columns, the implementation of this technology above commercial limits has been sparse due to difficulties in packing long columns and successfully operating instruments at ultrahigh pressures. In this work, a liquid chromatograph that operates up to 35 kpsi was investigated for the separation and identification of lipid species from human plasma. Capillary columns between 15-50 cm long were packed with 1.7 µm BEH C18 particles and evaluated for their ability to separate lipid isomers and complex lipid extracts from human plasma. Putative lipid class identifications were assigned using accurate mass and relative retention time data of the eluting peaks. Our findings indicate that longer columns packed and operated at 35 kpsi outperform shorter columns packed and run at lower pressures in terms of peak capacity and numbers of features identified. Packing columns with relatively high concentration slurries (200 mg/mL) while sonicating the column resulted in 6-34% increase in peak capacity for 50 cm columns compared to lower slurry concentrations and no sonication. For a given analysis time, 50 cm long columns operated at 35 kpsi provided a 20-95% increase in chromatographic peak capacity compared with 15 cm columns operated at 15 kpsi. Analysis times up to 4 h were evaluated, generating peak capacities up to 410 ± 5 (n = 3, measured at 4σ) and identifying 480 ± 85 lipids (n = 2). Importantly, the results also show a correlation between the peak capacity and the number of lipids identified from a human plasma extract. This correlation indicates that ionization suppression is a limiting factor in obtaining sufficient signal for identification by mass spectrometry. The result also shows that the higher resolution obtained by shallow gradients overcomes possible signal reduction due to broader, more dilute peaks in long gradients for improving detection of lipids in LC-MS. Lastly, longer columns operated at shallow gradients allowed for the best separation of both regional and geometrical isomers. These results demonstrate a system that enables the advantages of using longer columns packed and run at ultrahigh pressure for improving lipid separations and lipidome coverage.
在基于 LC-MS 的脂质组学中，样品制备、分离和质谱技术的改进不断扩大了其覆盖范围。虽然理论上，使用更长的、装填更小颗粒的色谱柱与使用更短的色谱柱相比，可以提供更高的分离性能，但由于在商业限制之上实现这项技术存在困难，包括长色谱柱的装填和在超高压力下成功运行仪器，因此该技术的应用仍然很少。在这项工作中，我们研究了一种能够在 35 kpsi 下运行的液相色谱仪，以分离和鉴定来自人血浆的脂质种类。我们使用 1.7 µm BEH C18 颗粒装填了 15-50 cm 长的毛细管柱，并评估了它们分离脂质异构体和复杂脂质提取物的能力。通过洗脱峰的精确质量和相对保留时间数据，对假定的脂质类别进行鉴定。我们的研究结果表明，在峰容量和鉴定特征数量方面，装填并在 35 kpsi 下运行的长色谱柱优于装填并在较低压力下运行的短色谱柱。使用相对高浓度的（200 mg/mL）浆料装填色谱柱，并对其进行超声处理，与较低的浆料浓度和不进行超声处理相比，50 cm 长的色谱柱的峰容量增加了 6-34%。对于给定的分析时间，与在 15 kpsi 下运行的 15 cm 长色谱柱相比，在 35 kpsi 下运行的 50 cm 长色谱柱的色谱峰容量增加了 20-95%。我们评估了长达 4 小时的分析时间，生成了高达 410 ± 5 的峰容量（n=3，在 4σ 处测量），并鉴定了 480 ± 85 种脂质（n=2）。重要的是，结果还显示了人血浆提取物的峰容量与鉴定的脂质数量之间的相关性。这种相关性表明，离子抑制是通过质谱进行充分鉴定的限制因素。结果还表明，在浅层梯度下获得的更高分辨率克服了长梯度中较宽、较稀释的峰导致的信号降低，从而改善了 LC-MS 中脂质的检测。最后，在浅层梯度下运行的长色谱柱能够实现区域异构体和几何异构体的最佳分离。这些结果证明了一种系统，该系统能够利用装填并在超高压力下运行的长色谱柱来提高脂质分离和脂质组学覆盖范围的优势。
