Phytochemical Analysis and Comparison between Controlled Environment Agriculture and Open-Field Growth of Spinach ( Spinacia oleracea L.) Using Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry

Ultrahigh-performance liquid chromatography–photodiode array–high-resolution tandem mass spectrometry (UHPLC-PDA-HRMS/MS n ) enabled the identification of 68 flavonoids and 29 other polar compounds in baby spinach cultivated in controlled environments and soil-based open-field production. Aglycone, glycosyl, and acyl substituents of each glycoside were deduced using diagnostic fragments from multiple stages of fragmentation. Putative compound assignments were made by comparing major fragments, UV absorption maxima, and the elution order with reference standards in an in-house database. Concentrations of key polyphenols were quantified using mole relative response factors (MRRF R ) referenced to rutin to enable comparison between production systems. Metabolite profiling revealed clear chemical differentiation between controlled environment agriculture (CEA) and open-field (OF) spinach. Several discriminating metabolites, including 5,3′,4′-trihydroxy-3-methoxy-6,7-methylenedioxyflavone 4′- O -glucuronide, jaceidin 4′- O -glucuronide, patuletin 3- O -pentosyldiglucoside, and spinacetin 3- O -pentosyldiglucoside, were highlighted as key spinach constituents, consistent with previous findings. This comprehensive phytochemical characterization of baby spinach showed clear shifts under different growing conditions, reflecting the sensitivity of secondary metabolism to environmental cues and the importance of growth conditions in shaping phytochemical profiles.