Accucore aq rp c18 column

Ten microliters of each sample were injected in a constrained randomized order into a Thermo Ultimate 3000 HPLC equipped with a Thermo Accucore aQ RP C18 column (100 × 2.1 mm, 2.6 µm particle size) and coupled to a Thermo Q-Exactive Orbitrap (all purchased from Thermo Fisher Scientific, Hägersten, Sweden). The mass spectrometer was operated in positive and negative ion mode and MS resolutions were set to 70,000 at m/z 200, AGC target 1,000,000, and maximum ion injection time 250 ms. A QC and a blank injection were done every eighth sample. Finally, a 2-fold serial dilution series ranging from 0.5 to 32.0 µL QC was injected. For improving metabolite identification, eight tandem mass spectrometry analyses in both ion modes were performed separately on pooled samples stratified on the diagnostic groups.

A detailed description of the metabolite extraction and mass spectrometry analysis is described in^{47 (link)}. In brief, samples were thawed on ice and 100 µL was mixed with ice-cold methanol spiked with a cocktail of internal standards. After vortexing and 30 min incubation in − 20 °C followed by centrifugation in 12 min at 4 °C, the samples were dried down and reconstituted in 100 µL 5% MeOH, 0.1% formic acid and 94.9% deionized MilliQ water upon analysis. 10 µL was injected in a randomized order constrained to the factor age into a Thermo Ultimate 3000 HPLC equipped with a Thermo Accucore aQ RP C18 column (100 × 2.1 mm, 2.6 µm particle size) and coupled to a Thermo Q-Exactive Orbitrap. A global pool of all samples was injected repeatedly, followed by a blank injection for quality control and filtering purposes. Finally, a twofold serial dilution series ranging from 0.5 to 32.0 µL QC was injected.