Reproducibility of non-fasting plasma metabolomics measurements across processing delays

Processing delays after blood collection is a common pre-analytical condition in large epidemiologic studies. It is critical to evaluate the suitability of blood samples with processing delays for metabolomics analysis as it is a potential source of variation that could attenuate associations between metabolites and disease outcomes.

We aimed to evaluate the reproducibility of metabolites over extended processing delays up to 48 h. We also aimed to test the reproducibility of the metabolomics platform.

Blood samples were collected from 18 healthy volunteers. Blood was stored in the refrigerator and processed for plasma at 0, 15, 30, and 48 h after collection. Plasma samples were metabolically profiled using an untargeted, ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) platform. Reproducibility of 1012 metabolites over processing delays and reproducibility of the platform were determined by intraclass correlation coefficients (ICCs) with variance components estimated from mixed-effects models.

The majority of metabolites (approximately 70% of 1012) were highly reproducible (ICCs ≥ 0.75) over 15-, 30- or 48-h processing delays. Nucleotides, energy-related metabolites, peptides, and carbohydrates were most affected by processing delays. The platform was highly reproducible with a median technical ICC of 0.84 (interquartile range 0.68-0.93).

Most metabolites measured by the UPLC-MS/MS platform show acceptable reproducibility up to 48-h processing delays. Metabolites of certain pathways need to be interpreted cautiously in relation to outcomes in epidemiologic studies with prolonged processing delays.