A comprehensive UHPLC-MS/MS and extraction method for spinach (Spinacia oleracea) flavonoids

Authors: Dzakovich, Michael; LE, ELAINE - Baylor College Of Medicine; TAK, ALVIN - Baylor College Of Medicine; CHACKO, SHAJI - Children'S Nutrition Research Center (CNRC)

Submitted to: bioRxiv
Publication Type: Rapid Release Publication
Publication Acceptance Date: 9/19/2024
Publication Date: 9/19/2024
Citation: Dzakovich, M.P., Le, E.A., Tak, A.L., Chacko, S.J. 2024. A comprehensive UHPLC-MS/MS and extraction method for spinach (Spinacia oleracea) flavonoids. bioRxiv. https://doi.org/10.1101/2024.09.13.612955.
DOI: https://doi.org/10.1101/2024.09.13.612955

Interpretive Summary: Flavonoids are bioactive compounds that are hypothesized to contribute to the health benefits associated with fruit and vegetable consumption. Spinach is a commonly consumed vegetable that contains a high concentration of these molecules. However, spinach flavonoids are not found in other fruits and vegetables and there are few methods available to quantify them. We developed a rapid (11.5 minutes/sample) analysis method that quantifies 39 spinach flavonoids even at extremely low concentrations. We also developed and evaluated a high-throughput extraction method that can be used to profile flavonoids in large populations of spinach. Our methodology gives plant biologists tools to understand how spinach flavonoid biosynthesis is controlled by genetic, environmental, and other factors. These tools allow for nutritional hypothesis about these compounds to be addressed opening new avenues of research.

Technical Abstract: Spinach 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. We 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. Our 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 through 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. Many 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.