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FTC does not exert the same effect on all metabolites. Although a large number of secondary metabolites were stable when subjected to five FTCs, FTC effects may lead to false-positive in the discovery of biomarker. In the case of reusing plant seed samples, no more than three consecutive freeze-thaw cycles were found advisable. This work provides unique perspectives on the FTC effects, which may fill in some existing gaps in the knowledge of the stability of plant metabolites during sample pre-handling.A bioanalytical method by high performance li