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An  in vivo  direct-immersion SPME sampling coupled to comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-ToFMS) was employed to capture real-time changes in the metabolome of ‘Honeycrisp’ apples during ripening on the tree. This novel sampling approach was successful in acquiring a broad metabolic fingerprint, capturing unique metabolites and detecting changes in metabolic profiles associated with fruit maturation. Several metabolites and chemical classes, including volatile esters, phenylpropanoid metabolites, 1-octen-3-ol, hexanal, and (2 E ,4 E )-2,4-hexadienal were found to be up-regulated in response to fruit maturation. For the first time, Amaryllidaceae alkaloids, metabolites with important biological activities, including anti-cancer, anti-viral, anti-parasitic, and acetylcholinesterase (AChE) inhibitory activity, were detected in apples. Considering the elimination of oxidative degradation mechanisms that adversely impact the representativeness of metabolome obtained  ex vivo , and further evidence that lipoxygenase (LOX) pathway contributes to volatile production in intact fruit,  in vivo  DI-SPME represents an attractive approach for global plant metabolite studies.

Application of in vivo solid phase microextraction (SPME) in capturing metabolome of apple (Malus ×domestica Borkh.) fruit