Urinary antihypertensive drug metabolite screening using molecular networking coupled to high-resolution mass spectrometry fragmentation

INTRODUCTION: Mass spectrometry is the current technique of choice in studying drug metabolism. High-resolution mass spectrometry in combination with MS/MS gas-phase experiments has the potential to contribute to rapid advances in this field. However, the data emerging from such fragmentation spectral files pose challenges to downstream analysis, given their complexity and size. OBJECTIVES: This study aims to detect and visualize antihypertensive drug metabolites in untargeted metabolomics experiments based on the spectral similarity of their fragmentation spectra. Furthermore, spectral clusters of endogenous metabolites were also examined. METHODS: Here we apply a molecular networking approach to seek drugs and their metabolites, in fragmentation spectra from urine derived from a cohort of 26 patients on antihypertensive therapy. The mass spectrometry data was collected on a Thermo Q-Exactive coupled to pHILIC chromatography using data dependent analysis (DDA) MS/MS gas-phase experiments. RESULTS: In total, 165 separate drug metabolites were found and structurally annotated (17 by spectral matching and 122 by classification based on a clustered fragmentation pattern). The clusters could be traced to 13 drugs including the known antihypertensives verapamil, losartan and amlodipine. The molecular networking approach also generated clusters of endogenous metabolites, including carnitine derivatives, and conjugates containing glutamine, glutamate and trigonelline. CONCLUSIONS: The approach offers unprecedented capability in the untargeted identification of drugs and their metabolites at the population level and has great potential to contribute to understanding stratified responses to drugs where differences in drug metabolism may determine treatment outcome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-016-1064-z) contains supplementary material, which is available to authorized users.