Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA; Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA.
J Chromatogr A. 2021 Jan 4;1635:461706. doi: 10.1016/j.chroma.2020.461706. Epub 2020 Nov 13.
LC-MS is an important tool for metabolomics due its high sensitivity and broad metabolite coverage. The goal of improving resolution and decreasing analysis time in HPLC has led to the use of 5 - 15 cm long columns packed with 1.7 - 1.9 µm particles requiring pressures of 8 - 12 kpsi. We report on the potential for capillary LC-MS based metabolomics utilizing porous C18 particles down to 1.1 µm diameter and columns up to 50 cm long with an operating pressure of 35 kpsi. Our experiments show that it is possible to pack columns with 1.1 µm porous particles to provide predicted improvements in separation time and efficiency. Using kinetic plots to guide the choice of column length and particle size, we packed 50 cm long columns with 1.7 µm particles and 20 cm long columns with 1.1 µm particles, which should produce equivalent performance in shorter times. Columns were tested by performing isocratic and gradient LC-MS analyses of small molecule metabolites and extracts from plasma. These columns provided approximately 100,000 theoretical plates for metabolite standards and peak capacities over 500 in 100 min for a complex plasma extract with robust interfacing to MS. To generate a given peak capacity, the 1.1 µm particles in 20 cm columns required roughly 75% of the time as 1.7 µm particles in 50 cm columns with both operated at 35 kpsi. The 1.1 µm particle packed columns generated a given peak capacity nearly 3 times faster than 1.7 µm particles in 15 cm columns operated at ~10 kpsi. This latter condition represents commercial state of the art for capillary LC. To consider practical benefits for metabolomics, the effect of different LC-MS variables on mass spectral feature detection was evaluated. Lower flow rates (down to 700 nL/min) and larger injection volumes (up to 1 µL) increased the features detected with modest loss in separation performance. The results demonstrate the potential for fast and high resolution separations for metabolomics using 1.1 µm particles operated at 35 kpsi for capillary LC-MS.
LC-MS 是代谢组学的重要工具，因为它具有高灵敏度和广泛的代谢物覆盖范围。提高 HPLC 分辨率和缩短分析时间的目标导致使用 5-15 厘米长的柱，填充 1.7-1.9µm 颗粒，需要 8-12kpsi 的压力。我们报告了基于毛细管 LC-MS 的代谢组学的潜力，利用直径为 1.1µm 的多孔 C18 颗粒和长达 50 厘米的柱，操作压力为 35kpsi。我们的实验表明，有可能用 1.1µm 多孔颗粒填充柱，以提供分离时间和效率的预期改善。使用动力学图来指导柱长和粒径的选择，我们用 1.7µm 颗粒填充 50 厘米长的柱，用 1.1µm 颗粒填充 20 厘米长的柱，这应该在更短的时间内产生等效的性能。通过对小分子代谢物和血浆提取物进行等度和梯度 LC-MS 分析来测试这些柱。这些柱对代谢物标准品提供了约 100000 个理论塔板，对于复杂的血浆提取物，在 100 分钟内的峰容量超过 500，并且与 MS 具有强大的接口。为了生成给定的峰容量，在 35kpsi 下，20 厘米长的 1.1µm 颗粒柱所需的时间约为 50 厘米长的 1.7µm 颗粒柱的 75%，而两者都在运行。用 1.1µm 颗粒填充的柱生成给定的峰容量的速度几乎是在 35kpsi 下运行的 1.7µm 颗粒填充的 15 厘米柱的 3 倍。后一种情况代表了毛细管 LC 的商业现状。为了考虑代谢组学的实际好处，评估了不同的 LC-MS 变量对质谱特征检测的影响。较低的流速（低至 700nL/min）和较大的进样量（高达 1µL）增加了检测到的特征，分离性能略有下降。结果表明，使用 35kpsi 操作的 1.1µm 颗粒在毛细管 LC-MS 中用于代谢组学具有快速和高分辨率分离的潜力。
