A Novel Method for Identification and Quantification of Sulfated Flavonoids in Plants by Neutral Loss Scan Mass Spectrometry

Abstract

Sulfur is present in plants in a large range of essential primary metabolites, as well as in numerous natural products. Many of these secondary metabolites contain sulfur in the oxidized form of organic sulfate. However, except of glucosinolates, very little is known about other classes of such sulfated metabolites, mainly because of lack of specific and quantitative analytical methods. We developed an LC-MS method to analyze sulfated flavonoids, a group of sulfated secondary metabolites prominent, e.g., in plants of the genus Flaveria . The method uses a linear gradient of methanol/formic acid in water on a Restek Raptor C 18 Core-Shell column for separation of the compounds. The sulfated flavonoids are detected by mass spectrometry (MS) in a negative mode, using a neutral loss of 80 Da after a collision induced dissociation. With this method we were also able to quantify the sulfated flavonoids. We could detect all (mono)sulfated flavonoids described before in Flaveria plus a number of new ones, such as isorhamnetin-sulfate-glycoside. In addition, we showed that sulfated flavonoids represent a substantial sulfur pool in Flaveria , larger than the thiols glutathione and cysteine. The individual species possess different sulfated flavonoids, but there is no correlation between the qualitative pattern and type of photosynthesis. Similar to other sulfur-containing secondary compounds, the concentration of sulfated flavonoids in leaves is reduced by sulfur starvation. The new LC-MS method will enable qualitative and quantitative detection of these secondary metabolites in plants as a pre-requisite to addressing their functions.

Combined Use of Endophytic Bacteria and Pre-Sowing Treatment of Thiamine Mitigates the Adverse Effects of Drought Stress in Wheat (Triticum aestivum L.) Cultivars

“…Chloroplast lipid peroxidation extent is estimated by measuring malondialdehyde (MDA) contents [48]. Flavonoids are well known low-molecular-weight secondary metabolites acting as antioxidants, anti-inflammatory, anti-mutagenic effects and plant pigmentation, which are proven to protect plants against drought stress [49]. A decrease in antioxidants by thiamine application (D-T3) signified it's imperative functioning towards alleviating drought stress.…”

Phytochemical Antioxidants: Past, Present and Future

“…Flavonoids act as inhibitors of the human sulfotransferases (SULTs) [66]. In plants, sulfate esters of flavonoids are rare compounds [77,78] that are found in species occurring coastal and swampy areas as well as arid habitats [78]. Functions of sulfated flavonoids in plants and animals are not clear [79].…”

Sulfation pathways from red to green

“…Sulfated flavonoids represent a large pool of sulfur in Flaveria. Indeed, in Flaveria pringlei 4.6% of sulfur can be found in quercetine 3-sulfate, and therefore 4-fold more than in GSH (170). The regulation of synthesis of sulfated flavonoids is completely unknown.…”

“…In addition to investigations at the systems level, newly developed technologies will certainly provide the cutting edge in sulfation pathway research. On the one hand, these are analytical nature-advanced methods to detect, characterize, and quantitate various sulfo-conjugates such as peptides (99), steroids (202), or plant secondary metabolites (170). On the other hand, these also represent novel chemo-synthetic approaches to obtain good purity sulfated nucleotides (203), steroid reference material (204), or sugars (205).…”