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 realtime 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 (2E,4E)-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 anticancer, 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.In food analysis, there is increasing interest in the implementation of metabolomics approaches to understand complex biological networks that control production of high-quality food commodities and crop plants, from both health-and safety-related aspects. In metabolomics applications, great care should be expended when selecting sample preparation and extraction methods for a given application, as the employed methods play a large role in determining metabolome coverage, and subsequently, the quality of the attained data 1 . In this regard, attainment of comprehensive metabolite coverage necessitates implementation of non-selective and unbiased sample preparation and extraction methods. Further, as an essential component of such methods, metabolomics sampling and sample preparation protocols must incorporate a suitable metabolism-quenching step to terminate enzymatic activity, prevent enzyme-mediated metabolite conversions, and eliminate chemical breakdown of labile metabolites. Metabolism quenching of plant tissues is traditionally carried out by methods such as freezing with liquid nitrogen, freeze-drying, and addition of alcohol or acid 2 . However, implementation of these traditional methods may result in alterations to the metabolomic profile due a multiplicity of factors, such as metabolite decompositions and interconversions, losses of volatile metabolites, emission of touch-or wound-induced metabolites, and non-reversible losses of metabolites by absorption to cell walls and membranes 3-6 . Hence, it becomes questionable whether the metabolome captured under such circumstances is a true signature of the biochemical activity of the investigated system.Given the importance of apples as a valuable food crop with high worldwide demand, several studies have taken as focus the profiling of volatile metabolites ...