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This study investigates the metabolic changes in leaf extracts of Nicotiana attenuata after simulated insect herbivory by mechanically wounding and applying oral secretions of Manduca sexta (W+OS) using HPLC-ESI-TOF-MS. Responses of wild-type (WT) plants are compared with genetically modified plants in which the hydroxyproline-rich glycopeptide systemin precursor (ppHypSys) is either over-expressed (OVsys) or down-regulated (IRsys). The challenges inherent to this type of study and strategies for quality control are discussed. Treated and untreated leaves (control samples) from WT, IRsys, and OVsys plants were harvested 1, 14, 86, and 120 h after treatment. The flash-frozen plant tissue was extracted with a MeOH/acetic acid buffer 40:60 (v/v). Individual extracts from five biological replicates per time-point, treatment and genotype were analyzed by HPLC-MS with a 45min binary acetonitrile-water gradient using an ESI-Qq-TOF for MS and MS/MS information for structure elucidation. Statistical analysis was performed using the Bruker ProfileAnalysis as well as XCMS and R software. Preliminary results obtained from a first data set of the same sample origin were used to design the experiment presented here. The number of biological replicates was increased from three to five to access the biological variation. The chromatographic separation was optimized in order to minimize suppression effects due to coelutions. A pooled sample generated from all individual samples was spiked with internal standards and served as quality control sample to monitor instrumental variation. The samples were analyzed in two different laboratories independently. The application of accurate and high resolution MS/MS data to support structure elucidation will be demonstrated.