Preparation of urine samples prior to targeted or untargeted metabolomics mass-spectrometry analysis

Steps preceding introduction of the analytical sample into individual separation equipment and/or mass-spectrometry (MS) detectors for targeted or untargeted metabolomics studies have not generally received the required attention, but particularly in dealing with urine samples. These steps have been classified into urine sample-pretreatment steps (i.e. those encompassing from sampling to storage) and urine sample-preparation steps (i.e. from thawing to insertion into the separation device and/or detector).We first discuss the steps constituting the former group (sampling time and collection, quenching, preservative addition, volume correction, pH adjustment, deproteinization – by centrifugation, normal filtration, ultrafiltration, ultrasound-assisted filtration and freezing/thawing cycles), and of those forming the latter group (dilution, enzymatic hydrolysis, sample clean-up and preconcentration – by solid-phase extraction, liquid–liquid extraction, evaporation, and lyophilization).We then reach conclusions that open a door to future, necessary research to improve the field of metabolomics on urine samples.

► We review steps preceding storage of urine samples, including sample preparation.
► We emphasize steps with most influence on targeted and untargeted analysis.
► We outline improvements of problematic steps in pre-treatment and sample preparation.