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 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 degrees C for 72 h. Results: the most effective protocol consisted of the combination between 200 mu L urease solution with 1-h sonication time; urease solution volumes > 200 mu L increase the risk to underestimate metabolite peaks because of sample dilution. Short-term storage of samples at -80 degrees 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 degrees C should be carefully reconsidered.