CSF samples were mixed with ethanol, vortexed, incubated (-20 °C) and centrifuged, as described by Song et al. . The supernatant was evaporated to dryness and reconstituted using Milli-Q water:MeOH:MeCN (2:1:1, v/v/v). Ten internal standards (IS) were added (Table ) and pooled Quality Control (QC) samples were prepared according to recent recommendations (SI, Section 1.2, Figure ). The IS (spiked in all CSF samples) were employed to check the instrument performance but were not included in the subsequent data analysis. The sample preparation method was first tested on artificial CSF samples (HelloBio Ltd, UK) using the same protocol as above, with the addition of 10 µL of a mixture containing 121 polar chemical standards (50 µM) to serve as reference standards later. Further details are given the SI (S1.3, Table and