Targeted metabolomics on malaria-infected mouse blood

We sampled mice in blocks (A-D), meaning each individual mouse was sampled every eight hours during the 26-hour sampling window, with 14 time points in total. We did not sample each mouse at each sampling point to minimise the total volume of blood being taken. We analysed blood plasma samples using the AbsoluteIDQ p180 targeted metabolomics kit (Biocrates Life Sciences AG, Innsbruck, Austria) and a Waters Xevo TQ-S mass spectrometer coupled to an Acquity UPLC system (Waters Corporation, Milford, MA, USA). We prepared the plasma samples (10 μl) according to the manufacturer’s instructions, adding several stable isotope–labelled standards to the samples prior to the derivatization and extraction steps. Using UPLC/MS (ultra performance liquid chromatography/mass spectrometry), we quantified 185 metabolites from 5 different compound classes (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, and sphingolipids). We ran the samples on two 96-well plates, randomised the sample order and ran three levels of quality control (QC) on each plate. We normalised the data between the plates using the results of quality control level 2 (QC2) repeats across the plate (n=4) and between plates (n=2) using Biocrates METIDQ software (QC2 correction).