Impact of preanalytical freezing delay time on the stability of metabolites in oral squamous cell carcinoma tissue samples

Introduction Metabolite stability is critical for tissue metabolomics. However, changes in metabolites in tissues over time from the operating room to the laboratory remain underexplored. Objectives In this study, we evaluated the effect of postoperative freezing delay time on the stability of metabolites in normal and oral squamous cell carcinoma (OSCC) tissues. Methods Tumor and paired normal tissues from five OSCC patients were collected after surgical resection, and samples was sequentially quenched in liquid nitrogen at 30, 40, 50, 60, 70, 80, 90 and 120 min (80 samples). Untargeted metabolic analysis by liquid chromatography–mass spectrometry/mass spectrometry in positive and negative ion modes was used to identify metabolic changes associated with delayed freezing time. The trends of metabolite changes at 30–120 and 30–60 min of delayed freezing were analyzed. Results 190 metabolites in 36 chemical classes were detected. After delayed freezing for 120 min, approximately 20% of the metabolites changed significantly in normal and tumor tissues, and differences in the metabolites were found in normal and tumor tissues. After a delay of 60 min, 29 metabolites had changed significantly in normal tissues, and 84 metabolites had changed significantly in tumor tissues. In addition, we constructed three tissue freezing schemes based on the observed variation trends in the metabolites. Conclusion Delayed freezing of tissue samples has a certain impact on the stability of metabolites. For metabolites with significant changes, we suggest that the freezing time of tissues be reasonably selected according to the freezing schemes and the actual clinical situation.