Non-target analysis and stability assessment of reference materials using liquid Chromatography‒High-Resolution mass spectrometry

Development and characterization of biological and environmental matrix certified reference materials (CRMs) for organic analytes typically relies heavily on targeted analytical methods, such as liquid chromatography (LC) with triple-quadrupole mass spectrometry detection. LC with high-resolution mass spectrometry (LC‒HRMS) can also provide high quality data for both targeted and non-targeted analytes, with the potential for retrospective data analysis. Here, we demonstrate the utility of non-target analysis (NTA) using LC‒HRMS for profiling and stability assessment of a mussel tissue matrix CRM certified for several classes of marine algal toxins (CRM-FDMT1). First, the NTA method was developed using data-dependent MS/MS acquisition and commercial metabolomics software for data processing. Of 128 toxin analogues previously reported in CRM-FDMT1, 125 were detected by LC-HRMS, with 97 triggered for MS/MS by data dependant acquisition. Automated data processing detected 119 of these compounds and 109 were retained after automated filtering of results for putative toxin analogues. Those analogues not detected were low abundance ions, or poorly resolved isomers. The method was then used to demonstrate new strategies for CRM stability assessment considering the stability of certified analytes, related toxin analogues, and unrelated matrix compounds. Several analogues from each toxin class in CRM-FDMT1 as well as other unrelated matrix compounds were observed to be significantly less stable than the certified toxins. Using this method, no instability was measured for any compounds at conditions ≤4 °C, providing a greater degree of confidence in CRM stability than could be achieved using conventional approaches to stability assessment targeting only the certified analytes. The NTA method and stability assessment approach presented are applicable to future CRM development with other matrices and organic analyte classes. • LC–HRMS/MS and data processing methods for non-target analysis of algal toxins in a matrix certified reference material. • Data processing workflow for CRM stability testing considers certified analytes, toxin analogues, and matrix components. • NTA in CRM stability testing enables retrospective analysis and non-target matrix stability.