La Gioia Danila, Salviati Emanuela, Basilicata Manuela Giovanna, Felici Claudia, Botrugno Oronza A, Tonon Giovanni, Sommella Eduardo, Campiglia Pietro
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 84084, Fisciano, SA, Italy.
PhD Program in Drug Discovery and Development, University of Salerno, Fisciano, SA, Italy.
Anal Bioanal Chem. 2025 May;417(13):2837-2847. doi: 10.1007/s00216-024-05588-z. Epub 2024 Oct 24.
Untargeted metabolomics UHPLC-HRMS workflows typically employ narrowbore 2.1-mm inner diameter (i.d.) columns. However, the wide concentration range of the metabolome and the need to often analyze small sample amounts poses challenges to these approaches. Reducing the column diameter could be a potential solution. Herein, we evaluated the performance of a microbore 1.0-mm i.d. setup compared to the 2.1-mm i.d. benchmark for untargeted metabolomics. The 1.0-mm i.d. setup was implemented on a micro-UHPLC system, while the 2.1-mm i.d. on a standard UHPLC, both coupled to quadrupole-orbitrap HRMS. On polar standard metabolites, a sensitivity gain with an average 3.8-fold increase over the 2.1-mm i.d., along with lower LOD (LOD 1.48 ng/mL vs. 6.18 ng/mL) and LOQ (LOQ 4.94 ng/mL vs. 20.60 ng/mL), was observed. The microbore method detected and quantified all metabolites at LLOQ with respect to 2.1, also demonstrating good repeatability with lower CV% for retention times (0.29% vs. 0.63%) and peak areas (4.65% vs. 7.27%). The analysis of various samples, in both RP and HILIC modes, including different plasma volumes, dried blood spots (DBS), and colorectal cancer (CRC) patient-derived organoids (PDOs), in full scan-data dependent mode (FS-DDA) reported a significant increase in MS1 and MS2 features, as well as MS/MS spectral matches by 38.95%, 39.26%, and 18.23%, respectively. These findings demonstrate that 1.0-mm i.d. columns in UHPLC-HRMS could be a potential strategy to enhance coverage for low-amount samples while maintaining the same analytical throughput and robustness of 2.1-mm i.d. formats, with reduced solvent consumption.
非靶向代谢组学超高效液相色谱-高分辨质谱工作流程通常采用内径为2.1毫米的窄孔柱。然而，代谢组的浓度范围很广，而且经常需要分析少量样本，这给这些方法带来了挑战。减小柱径可能是一个潜在的解决方案。在此，我们评估了内径为1.0毫米的微孔装置与内径为2.1毫米的非靶向代谢组学基准装置的性能。内径为1.0毫米的装置在微型超高效液相色谱系统上运行，而内径为2.1毫米的装置在标准超高效液相色谱上运行，两者均与四极杆-轨道阱高分辨质谱联用。在极性标准代谢物上，灵敏度提高，平均比内径为2.1毫米的装置增加3.8倍，同时检测限（检测限为1.48纳克/毫升，而6.18纳克/毫升）和定量限（定量限为4.94纳克/毫升，而20.60纳克/毫升）更低。微孔方法在最低定量限下检测和定量了所有相对于2.1的代谢物，保留时间（0.29%对0.63%）和峰面积（4.65%对7.27%）的变异系数更低，也证明了良好的重复性。在全扫描数据依赖模式（FS-DDA）下，对包括不同血浆体积、干血斑（DBS）和结直肠癌（CRC）患者来源的类器官（PDO）在内的各种样品进行反相（RP）和亲水相互作用液相色谱（HILIC）模式分析，结果显示母离子和子离子特征以及串联质谱谱图匹配分别显著增加了38.95%、39.26%和18.23%。这些发现表明，超高效液相色谱-高分辨质谱中内径为1.0毫米的柱可能是一种潜在策略，可在保持与内径为2.1毫米的柱相同的分析通量和稳健性的同时，提高对少量样品的覆盖范围，并减少溶剂消耗。
