Comparative evaluation of cerebral tissue pretreatment strategies for metabolomics using UHPLC-high-resolution mass spectrometry

Background The cerebrum, as the most complex human organ, contains diverse metabolites vital for understanding brain functions, diseases, drug effects, and addiction. Its liquefiable nature makes swift preparation crucial to preserve metabolic profiles, with physical homogenization needed to release metabolites. To determine the optimal preparation procedures for cerebrum samples for high-resolution mass spectrometry in metabolomics, we evaluated four homogenization techniques and three extraction protocols using UHPLC-HRMS. New method A combination of dry grinding and monophasic extraction was identified as an optimized sample preparation approach for cerebrum metabolomics. Results The number of feature peaks, coefficient of variation (CV) of peak areas, and peak areas were compared to evaluate the coverage, reproducibility, and sensitivity of the methods. The dry grinding method detected the most feature peaks among four methods of homogenization, and the peak areas obtained were superior to other homogenization methods. In terms of extraction methods, there was no significant difference in peak areas (except for tyrosine) between monophasic and two-step extraction methods (p > 0.05), both of which were higher than the biphasic extraction method (p < 0.05). Additionally, the reproducibility of the monophasic extraction method was significantly better than the other two methods. Comparison The dry grinding and monophasic extraction approach demonstrated excellent compatibility with UHPLC-HRMS, offering broader metabolite coverage and superior performance compared to existing methods. Conclusions The dry grinding and monophasic extraction method is recommended for cerebrum metabolomics, providing enhanced metabolite coverage, reproducibility, and adaptability for high-resolution mass spectrometry studies.