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

Abstract

BackgroundBefore 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. Methods92 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 30 min 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 72 h. Resultsthe most effective protocol consisted of the combination between 200 μL urease solution with 1-h sonication time; urease solution volumes >200 μL 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. Conclusionsour 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.