Proposed MS fragmentation pathway for chalcone derivatives. 

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... –ESI-MS-MS techniques. According to their chemical structures, UV absorption maxima and dominant fragmentation pathways, the standards could be classified into four types, including polymethoxylated flavones (Type A), flavanones (Type B), chalcones (Type C), and PMF glycosides (Type D). In the full scan mass spectra, all of them exhibited [M þ H] þ ions of sufficient abundance to be subsequently isolated and subjected to CID-MS-MS analysis ( Table I). The proposed fragmentation patterns were helpful to study the structures of PMFs. The nomenclature commonly used for mass fragments of flavonoids was adopted in this work (23). Two polymethoxylated flavone standards (Compounds P-1 and P-2) were analyzed first in the CID-MS-MS experiment. Comparing the product ion spectra of the standards (Figure 3), some characterized dissociation pathways could be summar- ized for further characterization of the other polymethoxylated flavones. First, all of their [M þ H] þ ions of standards could lose † one to four methyl radicals (CH 3 ) in their MS / MS spectra, and formed the base peaks of [M þ H–n  15] þ . This fragmentation pathway can be assumed as the major diagnostic characteristic for polymethoxylated flavones. Second, the other dissociation pathways by the loss of 16 (CH ), 18 (H O), 28 (CO), 31 (CH 4 CH 3 ), 33 (H 2 O CH 3 ), 43 (CO CH 3 ) and 61 † (CO þ H 2 O þ CH 3 ) were also frequently detected in the ESI-MS n spectra. These product ions could form their characteristic ESI-MS n fingerprint, which could be used to rapidly screen out the polymethoxylated flavones from the complex TCM system. The fragmentation pathways of two polymethoxylated flavanone derivatives (Compounds P-3 and P-4) were similar to each other in the CID-MS-MS experiment. P-4, for example, gave the [M þ H] þ ion at m / z 375, which further generated the base peak at m / z 221 in the MS spectrum (Figure 4). It could be deduced that its dominating fragmentation pathway was Retro-Diels-Alder (RDA) cleavage from the 1, 4-position of the C-ring. Meanwhile, the minor ion at m / z 181 was also detected in the result of the RDA fragmentation from the † 1, 3-position of the C-ring. The loss of 15 (CH 3 ), 28 (CO), † † 30 (2CH 3 ) and 31 (CH 3 þ CH 4 ) from the base peak at m / z 221 could also be detected as minor fragmentation ions in the CID-MS-MS spectra. The fragmentation pathway that the [M þ H] þ ion underwent in RDA reaction prior to the neutral loss of CH 3 , CH 4 , H 2 O and CO was noticeably different from ordinary flavanones Therefore, it could be adopted as a shortcut to rapidly distinguish them from ordinary flavones. Two polymethoxylated chalcone standards (Compounds P-5 and P-6) were also analyzed by the CID-MS-MS method. Their fragmentation pathways were similar to each other. Using P-5 as an example (Figure 5), the RDA cleavage at bond X to yield the base peak ion X B þ at m / z 221 and at bond Y to yield the minor ion A at m / z 211 (Figure 6) could also be simultaneously detected in the MS-MS spectrum. The fragmentation pathway was highly similar with flavanones. This is reasonable because the cyclization of 6’-hydroxychalcones to flavanones has been reported in many studies, which demonstrated that an intramolecular equilibrium is present between a flavanone- type and a chalcone-type of molecular ion (24, 25). Therefore, according to their fragmentation pathways, it was easy to distinguish polymethoxylated chalcones from polymethoxylated flavones, but difficult to distinguish them from polymethoxylated flavanones. However, the differences of UV absorption spectra between polymethoxylated chalcones and polymethoxylated flavanones provided an easy way to distinguish them, because the maximum absorption of chalcones usually ranged from 330 to 370 nm, whereas flavanones maintained at approximately 320 nm. Compounds P-7 and P-8, all attributed to PMF glycoside standards, were analyzed last by the CID-MS-MS method. First, their [M þ H] þ ions readily eliminated the sugar moiety to produce the corresponding [Aglycone þ H] þ ions as the base peaks in MS spectra. Next, the other dissociation pathways of [Aglycone þ H] þ by the loss of 15 (CH † 3 ), 18 (H 2 O), 30 (2CH † 3 ), 31 (CH † 3 þ CH 4 ), 33 (H 2 O þ CH † 3 ), 43 (CH † 3 þ CO) and 46 (H 2 O þ CO) were detected as diagnostic fragments in their MS n spectra, which were in accordance with the fragmentation pathways of polymethoxylated flavones (Figure 7). These primary product ions could also form the characteristic ESI-MS n fingerprint of PMF glycosides, which could be used to rapidly screen them out from the complex system. PMFs in the leaves of M. exotica have the basic aglycone structure with a maximum of seven substituents, such as a methoxyl group (OCH 3 ) and / or hydroxyl group (OH) on their A, B and C rings. The molecular weights of basic structures of aglycone are 222, 224 and 224 Da for flavones, flavanones and chalcones, respectively, which are increased by 30 or 16 Da when a methoxyl or hydroxyl is attached. On the basis of the numbers and types of substituent groups, the chemical formula and mass of every possible PMF isomer can be designated in advance (Tables II and III). Because of the complexity and similarity of PMFs in the leaves of M. exotica , the extracted ion chromatogram (EIC)-MS method was adopted to analyze the PMFs (Figure 8 and Table IV). The abundances of most of the unknown compounds, especially the chalcones and flavanones, were too low to use the online UV absorption spectra, so it was difficult to distinguish them from each other. Therefore, they were screened and identified together at last. After screening the molecular weights with the EIC-MS method, 36 candidates for PMFs were preliminarily screened from the leaves of M. exotica . However, the [M þ H] þ of 10 candidates (Peaks 1 –2, 21, 23, 25, 27, 30, 31, 34 and 36) have undergone completely different fragmentation pathways than those of PMF standards, so they were not characterized as PMFs. The other 26 candidates were tentatively identified as 18 polymethoxylated flavones, five flavanones or chalcones and three PMF glycosides (Table V) according to the diagnostic fragmentation pathways of the PMF standards. Among them, 24 PMFs were identified as OH-PMFs, whereas the rest were all permethoxylated PMFs. Meanwhile, the intensities of some EIC-MS peaks were too weak to be observed in the total ion chromatogram (TIC) spectra. Moreover, the retention times of some EIC-MS peaks were so similar that they could not be simultaneously identified in TIC spectra. However, the EIC method has been helpful to display every peak, especially the weak peaks in the highly complex mixtures. Thus, the EIC-MS method was confirmed to be powerful enough to preliminarily screen the constituents in highly complex TCM extracts. In the paper, a sensitive HPLC–DAD –ESI-MS-MS method was established that could be used to simultaneously identify and screen the PMFs present in the leaves of M. exotica . Eight PMF standards, including two flavones, two flavanones, two chalcones and two glycosides, were analyzed by CID-MS-MS to obtain the respective characteristics of fragment pathways, which could be used as the basis for further analysis of the PMFs in the extract. Meanwhile, owing to regularities of PMFs in elemental composition, the EIC-MS method by molecular weights was used to screen the homoeomorphic PMFs in its extract. In the end, 26 PMFs were identified preliminarily, including 23 PMFs and three PMF glycosides. This was the first systematic report on the presence of PMFs in the leaves of M. exotica , especially for polymethoxylated flavanones, polymethoxylated chalcones and PMF glycosides. The results indicated that the developed HPLC–DAD –ESI-MS-MS method could be employed as a rapid, effective technique to screen and identify PMFs from TCM ...