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 C₁₈ 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.

Keywords: Flaveria; mass spectrometry; method development; neutral loss scan; sulfated flavonoids; sulfur metabolism.

FIGURE 1

Structure of quercetin-3-sulfate. Sulfoflavonoids consist of the flavonoid specific polyphenolic C6-C3-C6 structure with one or more sulfate groups attached to the hydroxyl groups. The numbers refer to the carbon atoms and the positions of sulfations and other modifications.

FIGURE 2

Exemplary total ion chromatograms of six Flaveria species grown under control S conditions. Extracts were diluted 1:30 with methanol and the injection volume was 5 μl. Peaks are labeled with their retention times. The chromatograms relate to the Flaveria species (A) F. pringlei, (B) F. robusta, (C) F. linearis, (D) F. anomala, (E) F. australasica (F) F. bidentis. An enlarged view is given for regions with minor peaks in the chromatograms displayed in B–D.

FIGURE 3

Area ratios of putative isomers of sulfated isorhamnetin in six Flaveria species grown in control and low S conditions. (A) Area ratios of the plant samples grown in control S condition. (B) Area ratios of the plant samples grown under low S condition. Different colors represent different sulfated isorhamnetin isomers. Data are shown as mean values and standard deviation from 3 biological replicates.

FIGURE 4

Sulfated flavonoids affected by S starvation. Relative abundances of sulfated flavonoids with significant differences between control S (dark gray) and low S (light gray) grown Flaveria species. Data are shown as mean values and standard deviation from 3 biological replicates. Asterisks represent significance levels calculated by a two-sided student’s t-Test (^*p < 0.05; ^∗∗p < 0.005). Characters A and B in the x-axis labels indicate putative isomers with different retention times.

FIGURE 5

Absolute concentrations of sulfated quercetin in Flaveria species. Quercetin-3-sulfate was determined using the new LC-MS procedure in 6 Flaveria species grown at control (dark gray) or low S (light gray) supply. Data are shown as mean values and standard deviation from 3 biological replicates. Asterisks represent significance levels calculated by a two-sided student’s t-Test (^*p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005).

FIGURE 6

Partitioning of S in different pools in leaves of F. pringlei. Total S, sulfate, glutathione, cysteine, and sulfated quercetin were measured in leaves of F. pringlei. Data are shown as percentage of S in the individual compounds.

FIGURE 7

Score plot of the PCA model for the six Flaveria species grown under control S conditions. For the PCA the area ratios of all detected compounds were used. Each circle represents one individual sample.