Urine metabolome analysis by gas chromatography–mass spectrometry (GC–MS): Standardization and optimization of protocols for urea removal and short-term sample storage

Background: Before derivatization, urine analyzed by gas chromatography–mass spectrometry (GC–MS) requires the complete removal of urea to avoid interferences. We aimed at establishing the most effective sample pretreatment for urea removing; moreover, we explored the impact of two short-term sample storage conditions on urine metabolome. Methods: 92 aliquots were obtained from a single sample collected from a healthy adult; they were divided into 6 groups. Group 1 consisted of untreated aliquots while groups 2–6 differed from each other for the addition of various defined urease solution volumes combined with either 30min or 1-hour sonication time. Urine sample storage was tested by comparing 20 fresh aliquots analyzed after collection with 20 aliquots frozen at −80°C for 72h. Results: the most effective protocol consisted of the combination between 200μL urease solution with 1-h sonication time; urease solution volumes >200μL increase the risk to underestimate metabolite peaks because of sample dilution. Short-term storage of samples at −80°C pointed out significant changes in the urine metabolic profile compared with that of fresh samples. Conclusions: our study confirms the importance of urea removal for a reliable recognition and quantitation of metabolites; urine short-term storage at −80°C should be carefully reconsidered.