Mass Defect Filtering-Oriented Identification of Resin Glycosides from Root of Convolvulus scammonia Based on Quadrupole-Orbitrap Mass Spectrometer

This work aimed to develop and evaluate a post-acquisition data processing strategy, referred to as a mass defect filter (MDF), for rapid target the resin glycosides in root of Convolvulus scammonia by setting mass rang and mass defect range from high-resolution MS data. The full-scan mass data were acquired by high-performance liquid chromatography coupled with Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer that featured high resolution, mass accuracy, and sensitivity. To screen resin glycosides, three parent filter m/z 871, m/z 853, and m/z 869 combined with diagnostic fragment ions (DFIs) approach were applied to remove the interference from complex herbal extract. The targeted components were characterized based on detailed fragment ions. Using this approach, 80 targeted components, including 22 glycosidic acids and 58 resin glycosides were tentatively identified. The present results suggested that the proposed MDF strategy would be adaptable to the analysis of complex system in relevant filed.


Introduction
Convolvulus scammonia belongs to morning glory family (Convolvulaceae) and its extractive scammony, known commonly as resin glycosides which are unique secondary metabolites in natural. These active principles are responsible for the drastic purgative action and Convolvulaceous plants used in traditional medicine throughout the world, especially Convolvulus scammonia [1].
Resin glycosides are composed of differently acylated oligosaccharides glycosidically linked to hydroxylated fatty acids which are usually linked back to the sugar chain to form macro lactone rings of various sizes [1,9]. Even though phytochemical investigations on Convolvulus scammonia were initiated in the last nineteenth century, scammonic acid A together with scammonins I-VIII have been discovered [10][11][12], the structure of more active compounds still remain unrevealed. Due to the pivotal role in preventing and curing disease, the structural features of resin glycosides in Convolvulus scammonia should be investigated further. However, the structural complexity, high molecular weight, and lack of references render characterization and identification of resin glycosides challenging.

Construction of MDF Model
According to resin glycosides in Convolvulus scammonia were reported. Jalapinolic acid [(S)-11-hydroxyhexadecanoic acid] is the most frequently aglycone for the macrocyclic lipooligosaccharide core. Therefore, the parent drug filter was based on the jalapinolic acid, m/z 871 (C 40 H 71 O 20 ) picked as filter for glycosidic acids, m/z 853 (C 40 H 69 O 19 ) and m/z 869(C 40 H 69 O 20 ) for resin glycosides. Transformations such as 2-methylbutyric acid (Mba), tiglic acid (Tga), isobutyric acid (Iba), (2R,3R)-3-hydroxy-2-methylbutyric (Nia), 3-hydroxy-methylenebutyric (Hma) were supplied to MDF workflow. Using the mass defect values calculated above and the exact mass values calculated from the elemental composition input (Table 2), mass tolerance 112 Da and mass defect tolerance 0.058 Da were set to define the rectangular mass defect filters ( Table 3). The use of the MDF to filtrate targeted compounds from Convolvulus scammonia are given in Figure 3.

Construction of MDF Model
According to resin glycosides in Convolvulus scammonia were reported. Jalapinolic acid [(S)-11-hydroxyhexadecanoic acid] is the most frequently aglycone for the macrocyclic lipooligosaccharide core. Therefore, the parent drug filter was based on the jalapinolic acid, m/z 871 (C40H71O20) picked as filter for glycosidic acids, m/z 853 (C40H69O19) and m/z 869(C40H69O20) for resin glycosides. Transformations such as 2-methylbutyric acid (Mba), tiglic acid (Tga), isobutyric acid (Iba), (2R,3R)-3-hydroxy-2-methylbutyric (Nia), 3-hydroxy-methylenebutyric (Hma) were supplied to MDF workflow. Using the mass defect values calculated above and the exact mass values calculated from the elemental composition input (Table 2), mass tolerance 112 Da and mass defect tolerance 0.058 Da were set to define the rectangular mass defect filters ( Table 3). The use of the MDF to filtrate targeted compounds from Convolvulus scammonia are given in Figure 3.   The generated chromatograms are displayed in Figure 4. As shown in the chromatogram, after filtration by MDF technique, the noise level was lower than the original chromatogram and ions which the mass defect were not within the filter ranges, were The generated chromatograms are displayed in Figure 4. As shown in the chromatogram, after filtration by MDF technique, the noise level was lower than the original chromatogram and ions which the mass defect were not within the filter ranges, were excluded, the targeted components became predominant ions in the TIC. Meanwhile, the sensitivity of minor constituents such as m/z 1170.60286 at 59.46 min was largely improved.

Identified Glycosidic Acids
Glycosidic acids exhibited deprotonated ions as the base peak were observed; neutral loss of monosaccharide units including deoxyhexose, hexose and short organic acid moieties such as tiglic acid, 2-methylbutyric acid, (2R,3R)-3-hydroxy-2-methylbutyric were the main fragment patterns; aglycone ions m/z 257, m/z 271, m/z 285 indicated the presence of the hydroxylated fatty acid with chain lengths of C15, C16, and C17, respectively. A total of 22 glycosidic acids were detected and tentatively characterized in root of Convolvulus scammonia, in which 21 compounds were based on scammonic acid A parent filter, Their ESI-MS n information is shown in Table 4. Notable ion at m/z 1170 was founded that scammonic acid B as its parent drug. Diagnostic fragment ion at m/z 271 was the common aglycone, suggesting the presence of jalapinolic acid.

Identified Glycosidic Acids
Glycosidic acids exhibited deprotonated ions as the base peak were observed; neutral loss of monosaccharide units including deoxyhexose, hexose and short organic acid moieties such as tiglic acid, 2-methylbutyric acid, (2R,3R)-3-hydroxy-2-methylbutyric were the main fragment patterns; aglycone ions m/z 257, m/z 271, m/z 285 indicated the presence of the hydroxylated fatty acid with chain lengths of C 15 , C 16 , and C 17 , respectively. A total of 22 glycosidic acids were detected and tentatively characterized in root of Convolvulus scammonia, in which 21 compounds were based on scammonic acid A parent filter, Their ESI-MS n information is shown in Table 4. Notable ion at m/z 1170 was founded that scammonic acid B as its parent drug. Diagnostic fragment ion at m/z 271 was the common aglycone, suggesting the presence of jalapinolic acid.    Peaks listed below were all based on parent template m/z 853, totally 56 peaks were tentatively characterized, the detail of ESI-MS n information is shown in Table 5. Analysis of the MS/MS spectrum, the proposed fragmentation patterns of resin glycosides are as follows: