# Antioxidant Activity of Sulfate Metabolites of Chlorogenic Acid
## 3 Results  
### Characterization of Sulfate Derivatives of Ferulic, Caffeic, Dihydroferulic and Dihydrocaffe Acids  
Three main peaks were observed on the total ion chromatogram (TIC) obtained for the mixture after synthesis of the ferulic acid sulfate ester (Figure 2A). Peak 1 (P1), with a retention time of 12.8 min, produced a negatively charged pseudomolecular ion, [M-H]\({}^{-}\), at \(m/z=273\), corresponding to the value expected for ferulic acid-4-O-sulfate. The major fragment of the analyzed compound produced an ion at \(m/z=193\) characteristic of ferulic acid. A loss of 80 atomic mass units (amu) indicates cleavage of the sulfate moiety. Fragmentation ions produced in linear ion trap (MS3) with \(m/z=178\) and 149 were characteristic for fragmentation pathway of a standard of ferulic acid (Figure 2C). The retention time (14.8 min) and SRM pairs of the second peak (P2) were characteristic of ferulic acid, which was the main substrate used for the synthesis. The last peak (P3) was identified as an impurity. During the purification process, only the P1 fraction (10-13 min) was collected and re-analyzed using HPLC with mass detection. A final LC-MS/MS analysis allowed us to obtain a pure compound with a retention time of 12.8 min and fragmentation spectra characteristic of ferulic acid-4-O-sulfate (Figure 2B). We identified dihydroferulic acid-4-O-sulfate using a similar procedure. The TIC presents six main peaks (Figure 3A). The fraction between 9 and 13 min (P3) had [M-H]\({}^{-}\) at \(m/z=275\), which is characteristic of the expected product. The resulting MS2 fragmentation ion at \(m/z=175\) indicates a loss of 80 amu and is characteristic of dihydroferulic acid standards. Further fragmentation in linear ion trap (MS3) resulted in the presence of ions at \(m/z=151\), 135 of which are similar to that obtained for the substrate used. The fragmentation pathway of the synthesized product was in good agreement with previous studies and  
Figure 2: (**A**) TIC of mixture after synthesis of ferulic acid-4-O-sulfate; (**B**) chromatogram of ferulic acid-4-O-sulfate obtained after purification; (**C**) fragmentation spectra of ferulic acid-4-O-sulfate. expected fragmentation spectra (Figure 3C). The retention time (13.7 min), characteristic SRM pair and fragmentation pathway of peak 4 (P4) are characteristic of pure substrate-dihydrofervilic acid, which was used in the synthesis. The rest peaks were identified as impurities. Collection of only the P3 fraction allowed us to obtain a pure compound with a retention time of 12.0 min and fragmentation spectra characteristic of dihydrofervilic acid-4-\(O\)-sulfate (Figure 3B). Figure 4A presents the TIC of the mixture obtained after sulfation of caffeic acid. Peak 1 (P1) produced a negatively charged pseudomolecular ion [M-H]\({}^{-}\) at \(m/z=259\), which on MS2 produced fragmentation ion at \(m/z=179\) corresponding to the caffeic acid moiety. The ion produced on MS3 at \(m/z=135\) is characteristic of the fragmentation of caffeic acid. The fragmentation spectra of peak 1 (P1) correspond to the expected product, a sulfate ester of caffeic acid (Figure 4C). Two other peaks were identified as impurities. Contrary to the TIC obtained for mixtures after the synthesis of ferulic acid-4-\(O\)-sulfate and dihydrofervilic acid-4-\(O\)-sulfate, we did not observe a peak corresponding to a caffeic acid substrate. The purification process consisted of the collection of only the fraction between 8 and 12 min (P1). Re-analysis of the collected fraction allowed us to identify two products with similar fragmentation pathways but different retention times: 10.97 and 11.80 min (Figure 4B). The loss of 80 amu, which is characteristic of sulfate moieties, and MS3 producing an ion at \(m/z=135\) indicate that both peaks are sulfate esters of caffeic acid. Therefore, the obtained products correspond to two monoesters of caffeic acid (caffeic acid-4-\(O\)-sulfate and caffeic acid-3-\(O\)-sulfate). However, disulfate esters of caffeic acid were not observed.