Absoluteidq p180 targeted metabolomics kit

Plasma samples were measured 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) similar to our previous studies^{42 (link)–44 (link)}. Plasma samples (10 μl) were prepared according to the manufacturer’s instructions adding several stable isotope–labelled standards to the samples prior to the derivatization and extraction steps. Using either LC/MS or flow injection analysis/MS up to 183 metabolites from 5 different compound classes (namely acylcarnitines, amino acids, biogenic amines, glycerophospholipids and sphingolipids) can be quantified. All the plasma samples and 3 levels of quality control (QC) were processed on a single 96-well plate, sample order being randomised. The levels of metabolites present in each QC were compared to the expected values and the percent coefficient of variation (CV%) calculated. Metabolites where >25% concentrations were below the limit of detection (LOQ) or blank out of range, or the QC2 coefficient of variance was >30%, were excluded (n = 50)^{43 ,44 (link)}. The remaining 133 quantified metabolites comprised 10 acylcarnitines, 21 amino acids, 12 biogenic amines, 76 glycerophospholipids and 14 sphingolipids.

Plasma melatonin and cortisol concentrations were measured by radioimmunoassay (Stockgrand Ltd, University of Surrey) as described previously (Sletten, Revell, Middleton, Lederle, & Skene, 2009). Targeted LC/MS was performed on two‐hourly plasma samples to identify and quantify metabolite concentrations, 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) as previously described (Davies et al., 2014; Isherwood, Van der Veen, Johnston, & Skene, 2017; Skene et al., 2017). The kit provides absolute concentrations of 184 metabolites from six different compound classes (acylcarnitines, amino acids, biogenic amines, lysophosphatidylcholines, glycerophospholipids and sphingolipids). Plasma samples were prepared according to the manufacturer's instructions. Sample order was randomised and three levels of quality controls (QC) were run on each 96‐well plate. Data were normalised between batches using the QC level 2 (QC2) repeats across each plate (n = 4) and between plates (n = 5) using Biocrates METIDQ software (QC2 correction). Metabolites where >25% concentrations were below the limit of detection (LOQ) or blank out of range, or the QC2 coefficient of variance was >30%, were excluded (n = 54).

Metabolites were measured using the AbsoluteIDQ p180 targeted metabolomics kit (Biocrates Life Sciences AG, Innsbruck, Austria) which covers 6 classes of metabolites including amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins, on a Waters Xevo TQ-S mass spectrometer coupled to an Acquity H-Class LC system (Waters Corporation, Milford, MA, USA).