The composition and the role of each type of these materials is summarised in Table , and their integration with the sample handling and preparation workflow is illustrated in Figure . Method Reference (MR) mixtures are method-specific sets of reference chemicals not expected to be present in the biological samples being studied (i.e., heavy labelled small molecules and non-endogenous odd-chain lipids). Each MR is specific to a chromatographic method (small molecule RPC, HILIC and lipid RPC) and formulated for use with both ionization modes, where applicable. The MR mixture is added to all pooled QC samples providing metabolite targets that represent the wider observable metabolome while facilitating a more real-time assessment of data quality. To allow assessment of sample preparation and injection precision as well as some limited assessment of matrix effect across individual study samples, internal standards (IS) are added to all pooled QC and study samples. When preparing samples for small molecule RPC or HILIC profiling, MS and IS mixtures are prepared in aqueous solutions and added to the sample prior to further preparative steps. For lipidomic profiling, a combined MR/IS mixture (referred to as LipidMix in the lipid method protocol) is added to all samples during protein precipitation with 2-propanol owing to the inability to solubilise and add them in an aqueous solution prior. The chemical composition of each MR and IS, the ions routinely monitored, and their method-specific retention times are reported in Table . To facilitate quality control and assessment on the scale of the true chemical complexity of biological samples, two types of biological pooled QC samples are used for global profiling studies. The first, a study reference (SR), is a pooled QC sample composed of equal parts of all samples in the study except any purposefully excluded. Here the SR is used in the traditional sense for assessing the reproducibility of feature intensity measurement. Furthermore, by diluting the SR across a range of concentrations to form a set of "dilution series" QC samples, features can also be assessed for how well they are captured with linear dynamic range of the instrument, and for the proportionality of their response to dilution. The SR also forms the basis of pre-processing steps such as batch and run-order correction (see data extraction and pre-processing methods section below).