The Impact of the Serum Extraction Protocol on Metabolomic Profiling Using UPLC-MS/MS and FTIR Spectroscopy

Tiago A H Fonseca; Cristiana P Von Rekowski; Rúben Araújo; M Conceição Oliveira; Gonçalo C Justino; Luís Bento; Cecília R C Calado

doi:10.1021/acsomega.3c01370

The Impact of the Serum Extraction Protocol on Metabolomic Profiling Using UPLC-MS/MS and FTIR Spectroscopy

ACS Omega. 2023 Jun 1;8(23):20755-20766. doi: 10.1021/acsomega.3c01370. eCollection 2023 Jun 13.

Authors

Tiago A H Fonseca¹, Cristiana P Von Rekowski¹, Rúben Araújo¹, M Conceição Oliveira², Gonçalo C Justino², Luís Bento^{3

4}, Cecília R C Calado^{1

5}

Affiliations

¹ Instituto Superior de Engenharia de Lisboa (ISEL), Instituto Politécnico de Lisboa, Rua Conselheiro Emídio Navarro 1, 1959-007 Lisboa, Portugal.
² Centro de Química Estrutural, Institute of Molecular Sciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa, Portugal.
³ Intensive Care Department, Centro Hospitalar Universitário de Lisboa Central (CHULC), Rua José António Serrano, 1150-199 Lisboa, Portugal.
⁴ Integrated Pathophysiological Mechanisms, CHRC, NOVA Medical School, Faculdade de Ciências Médicas, NMS, FCM, Universidade NOVA de Lisboa, Campo Mártires da Pátria, 130, 1169-056 Lisboa, Portugal.
⁵ Centro de Investigação em Modelação e Optimização de Sistemas Multifuncionais (CIMOSM), Instituto Superior de Engenharia de Lisboa (ISEL), Instituto Politécnico de Lisboa, Rua Conselheiro Emídio Navarro 1, 1959-007 Lisboa, Portugal.

Abstract

Biofluid metabolomics is a very appealing tool to increase the knowledge associated with pathophysiological mechanisms leading to better and new therapies and biomarkers for disease diagnosis and prognosis. However, due to the complex process of metabolome analysis, including the metabolome isolation method and the platform used to analyze it, there are diverse factors that affect metabolomics output. In the present work, the impact of two protocols to extract the serum metabolome, one using methanol and another using a mixture of methanol, acetonitrile, and water, was evaluated. The metabolome was analyzed by ultraperformance liquid chromatography associated with tandem mass spectrometry (UPLC-MS/MS), based on reverse-phase and hydrophobic chromatographic separations, and Fourier transform infrared (FTIR) spectroscopy. The two extraction protocols of the metabolome were compared over the analytical platforms (UPLC-MS/MS and FTIR spectroscopy) concerning the number of features, the type of features, common features, and the reproducibility of extraction replicas and analytical replicas. The ability of the extraction protocols to predict the survivability of critically ill patients hospitalized at an intensive care unit was also evaluated. The FTIR spectroscopy platform was compared to the UPLC-MS/MS platform and, despite not identifying metabolites and consequently not contributing as much as UPLC-MS/MS in terms of information concerning metabolic information, it enabled the comparison of the two extraction protocols as well as the development of very good predictive models of patient's survivability, such as the UPLC-MS/MS platform. Furthermore, FTIR spectroscopy is based on much simpler procedures and is rapid, economic, and applicable in the high-throughput mode, i.e., enabling the simultaneous analysis of hundreds of samples in the microliter range in a couple of hours. Therefore, FTIR spectroscopy represents a very interesting complementary technique not only to optimize processes as the metabolome isolation but also for obtaining biomarkers such as those for disease prognosis.