Danila La Gioiaa,b, Eduardo Sommellaa, Pietro Campigliaa

aDepartment of Pharmacy, University of Salerno, Fisciano (SA), Italy;
bPhD Program in Drug Discovery and Development, University of Salerno, Fisciano (SA), Italy;

Untargeted metabolomics is invaluable for understanding disease mechanisms and treatment responses. While narrowbore LC-HRMS (2.1 mm i.d. columns) is common, higher sensitivity is needed for low-volume samples. Microbore columns (1.0 mm i.d.) have potential but are underutilized in metabolomics. We developed a sensitive microbore UHPLC-HRMS method for metabolomics, maintaining robustness, demonstrated using human plasma, Dried Blood Spots (DBS) and organoids. 

All the analyses were performed on a nano/microflow Vanquish Neo UHPLC coupled to an Orbitrap Exploris 120 mass spectrometer. Both RPLC and HILIC chromatography were performed with sub-2 micrometer fully porous particles 1.0 mm i.d. columns. UPCLC-HRMS analyses were performed in data dependent acquisition (DDA), using a resolution of 60000 in MS1 and 15000 in MS2, both positive and negative electrospray ionization were employed. Human plasma, DBS and organoids were used as test samples. Data alignment, filtering and metabolite annotation was performed with Compound Discoverer 3.0. Comparative analyses on 2.1 mm i.d. column was performed on a Vanquish Flex system coupled to a Orbitrap Exploris 120 MS. 

Flow rate, gradient, injection volume, column temperature and source parameters were investigated by a mixture of metabolites with different retention and ionization behavior. A flow rate of 100 μL/min resulted to be the best condition among those tested. Microbore UHPLC–HRMS shows excellent reproducibility of chromatographic retention time (≤0.5% coefficient of variation, CV) and peak area (≤2.3% CV), together with sharp and symmetrical peaks with an average Full Width at Half Maximum (FWHM) of 0.05 min. Additionally, the microbore approach showed higher sensitivity in full scan (FS)-DDA, resulting in LOD and LOQ values of 0.95 and 3.18 ng/mL. Compared to narrowbore method, a ∼2-fold increase in response was obtained on the same sample, this turns in almost twice MS1 spectral features and metabolite annotations at MSI level 2. The system robustness was proved in 48 hours and more than 300 consecutive injections. Notably, a reduction of solvent consumption is also obtained (300 mL vs 1.5L), making this approach more environmentally sustainable. As proof of concept, the microbore LC-HRMS method successfully profiled CRC organoids' metabolome, revealing modulations in several metabolic pathways following chemotherapy. 

Our findings underscore the robustness, sensitivity, and broad applicability of microbore UHPLC-HRMS in metabolomics, offering a promising alternative to conventional 2.1mm i.d. column-based approaches.

Keywords: Mass spectrometry, metabolomics, microbore, UHPLC, untargeted