The MALDI-TOF MS Of Vegetable Tannins

Convenient, simple and reliable methods to determine vegetable tannins structure is a prerequisite to study their numerous important properties in addition to the important role of tannins in ecological processes, including strong pharmacology and physiology activity, significant impacts on human and animal health, and their potential applications. Matrix-assisted Laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) provides a rapid and ideally method for characterization of polydispersed vegetable tannins. In the present study, three aspects 1) negative effects of fragmentation of tannins molecules in MALDI, 2) preparation processes for vegetable tannins on reliable determination of their chemical structure, and 3) MALDI-TOF MS combining with ¹³C-NMR for charactering the unknown specie of vegetable tannins were cautiously investigated.1. The oligomers of hydrolysable tannins are more breakable than thoseflavan-3-ol oligomers of condensed tannins in MALDI. The cationization reagents affect the MALDI-TOF MS spectrum of Chinese gallotannins significantly. MALDI-TOF of the Chinese gallotannins showed different series of peaks in the presence of different cations at the time of the MALDI. 1) When Cs⁺ was used as the cationization reagent, Chinese gallotannins gave a relatively simple MALDI-TOF spectrum, three series of quasimolecular ions [M + Cs]⁺, [M + 2Cs - H]⁺ and [M + 3Cs - 2H]⁺ and a series of metastable ions peaks with minimum abundance were detected. 2) Selection of Na⁺ as the cationization reagent, additional three series of ions peaks including two patterns from the fragmentation and complex 2M adducts [2M + Na]⁺ can be distinguished. 3) In the case of no deionization or addition of cationization reagent to the analyte /matrix, naturally abundant Na⁺ and K⁺ as the cationization reagent.2. In MALDI-TOF MS of condensed tannins, selection of Cs⁺ as the cationization reagent rather than Na⁺, we could observe 1) the condensed tannin polymers of higher polymerization degree (PD), 2) higher PD of the oligomers with the highest intensity ion peak, and 3) the higher average PD of the same condensed tannins. These were most likely to result from the severe fragmentation of flavan-3-ol oligomers and polymers when Na⁺ presented at the time of MALDI. The hydroxyl pattern of the condensed tannins (gallocatechin/epigallocatechin (GC/EGC) units in condensed tannins) detected by MALDI-TOF MS in the case of both Na⁺and Cs⁺as the cationization reagent were not significantly different. However, because of the naturally abundant K⁺, when Na⁺ was employed as the cationization reagent, the authentic results of hydroxyl pattern greatly depended on the degree of deionization. The strong cation-exchange resin should be selected as the deionization reagent to remove the extra cations from analyte and matrix (DHB) rather than the weak cation-exchange resin.3. If the average DP and distribution of oligomers and polymers wascalculated according to the abundance of molecular ions peaks or isotopic peaks with most intensity in isotopic clusters of oligomers and polymers in MALDI-TOF spectrum of vegetable tannins, the composition of polymers would be under-evaluated. These calculations would result in mass discrimination.4. To quantify the content of vegetable tannins in plant tissues or characterizetheir authentic chemical structure by MALDI-TOF, the 70% acetone solvent should be used to extract the almost of free tannins in fresh plant samples. Although the aqueous fraction containing tannins can be purified by liquid/liquid extraction with different solvents to remove lipid, chlorophyll and monomer phenols, the EtOAc as the extraction solvent will lead to unauthentic structure properties of vegetable tannins.The MALDI-TOF of condensed tannins indicated that the composition of GC/EGC units in condensed tannins was increased by the three tannin purification methods (method 1, Sephadex LH-20 sorbs tannins and nontannins fraction is washed by 50% methanol solution (50%MP), method 2, Sephadex LH-20 sorbs tannins and nontannins fraction is washed by ethanol (100%EP), and releases them in 70% acetone solution, and method 3, Yb³⁺ selectivity precipitates tannins). The flavan-3-ol polymers were partly lost from the purified condensed tannin by 50%MP. In addition to loss of polymers, the most oligomers (dimmer～hexamer) were lost from the purified condensed tannin by 100%EP, which would interfere with reliable determination of vegetable tannins structure.5. MALDI-TOF MS combining with ¹³C-NMR was successfully applied tocharacterize the unknown specie of condensed tannins extracted from the mature leaves of Ficus microcarpa L. The condensed tannins extracted from the leaves of Ficus microcarpa L. was predominantly composed of propelargonidin (PP), and were most homopolymers consisting of afzelechin/epiafzelechin units. In these condensed tannins, the epiafzelechin and afzelechin of PP were the primary units, reached 72.9% and 24.0% relative to total flavan-3-ol units respectively, and the catechin/epicatechin of procyanidin is just 3.3%. The terminal units were mainly 2, 3-cis stereochemistry, and contained only 6.5% 2, 3-trans stereochemistry units. The average DP was 4.9 detected by MALDI-TOF MS. In addition to a small quantity of galloyl flavan-3-ol oligomers, the flavan-3-ol oligomers and polymers containing glucoside observed slightly in the MALDI-TOF of condensed tannins extracted from mature leaves of Ficus microcarpa L.