AUTHOR=Dzakovich Michael P. , Le Elaine A. , Tak Alvin L. , Chacko Shaji K. 

TITLE=A comprehensive UHPLC-MS/MS and extraction method reveals flavonoid profile and concentration are diverse in spinach (Spinacia oleracea L.)

JOURNAL=Frontiers in Plant Science

VOLUME=Volume 16 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2025.1496200

DOI=10.3389/fpls.2025.1496200

ISSN=1664-462X

ABSTRACT=IntroductionSpinach produces an array of unique flavonoids not commonly found in other fruits and vegetables. These molecules likely serve as defense agents against biotic and abiotic stress and may have health beneficial properties for humans. Current methods to analyze spinach flavonoids are incomplete and only capture a portion of this uncharacterized pathway. A comprehensive analysis method is needed to determine how genetics, environmental conditions, and other factors influence spinach flavonoid biosynthesis.MethodsWe developed and validated a high-throughput extraction and ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to separate and quantify 39 spinach flavonoid species in 11.5 minutes. Spinach flavonoids without authentic standards were putatively identified using MS/MS fragmentation experiments, precursor scans, and matches to high-resolution MS literature reports.ResultsOur extraction method enables up to 48 samples to be extracted in 60 minutes with recovery estimates between 100.5 – 107.8%. To assess the suitability of our method and generate benchmark estimates for 39 spinach flavonoids, we grew a panel of 30 genetically diverse spinach accessions and compared quantification data generated with a traditional or our high-throughput approach. Data generated by either approach were comparable, estimating total flavonoid averages of 75.1 – 170.1 or 93.1 – 187.26 mg/100 g fresh weight for the high-throughput and traditional method, respectively.DiscussionMany estimates generated by our analysis method represent the first quantitative literature reports of these compounds. These experiments indicate that our extraction and analysis method is efficient, robust, and an important tool needed to study the biosynthesis and biological role of spinach flavonoids.