Table 1.
Molecular formulae, elemental changes relative to BTX-2,m/zof protonated ions, ring double bond equivalents (RDBE), retention times (RT), and tentative assignments for relevant compounds in this study. Listed accurate m/z values and RT for each compound were obtained from a sample collected after 60 min incubation of BTX-2 (5 μM, v/v, after dilution in the assay) with L. campechanus (RSN1), A. probatocephalus (SHP), or human (HLM) liver microsomes.
| Compound No. | Neutral Formula | Change relative to BTX-2 | Accurate [M+H]+m/z (Δm in ppm) | Ion RDBE | RTa (min) | Tentative Assignment |
|---|---|---|---|---|---|---|
| 1 | C50H70O14 | n/a | 895.4864 (+2.9) | 15.5 | 11.2 | BTX-2b |
| 2a | C50H72O14 | +H2 | 897.5030 (+3.9) | 14.5 | 8.3 | BTX-3b |
| 2b | C50H72O14 | +H2 | 897.5030 (+3.9) | 14.5 | 11.2 | 41,43-dihydro-BTX-2 |
| 3 | C50H74O14 | +H4 | 899.5183 (+3.5) | 13.5 | 8.5 | BTX-9 |
| 4a | C50H70O15 | +O | 911.4820 (+3.6) | 15.5 | 5.7 | Hydroxyl BTX-2 (C-9 or D-ring) |
| 4b | C50H70O15 | +O | 911.4828 (+4.4) | 15.5 | 7.5 | Hydroxyl BTX-2 (rings A-E) |
| 4c | C50H70O15 | +O | 911.4822 (+3.8) | 15.5 | 9.1 | BTX-B5b |
| 5a | C50H72O15 | +H2O | 913.4974 (+3.3) | 14.5 | 3.7 | |
| 5b | C50H72O15 | +H2O | 913.4980 (+3.9) | 14.5 | 5.1 | |
| 5c | C50H72O15 | +H2O | 913.4979 (+3.8) | 14.5 | 5.6 | A-ring hydrolyzed BTX-2b |
| 5d | C50H72O15 | +H2O | 913.4970 (+2.8) | 14.5 | 8.5 | 41,43-dihydro-BTX-B5c |
| 5e | C50H72O15 | +H2O | 913.4974 (+3.3) | 14.5 | 8.7 | 41,43-dihydro-BTX-B5c |
| 6a | C50H74O15 | +H4O | 915.5139 (+4.3) | 13.5 | 3.6 | A-ring hydrolyzed BTX-3b |
| 6b | C50H74O15 | +H4O | 915.5141 (+4.5) | 13.5 | 5.3 | |
| 7a | C50H70O16 | +O2 | 927.4769 (+3.5) | 15.5 | 4.1 | |
| 7b | C50H70O16 | +O2 | 927.4768 (+3.4) | 15.5 | 5.7 | |
| 8 | C50H72O16 | +H2O2 | 929.4920 (+2.9) | 14.5 | 4.2 | A-ring hydrolyzed BTX-B5b |
| 9 | C51H76O15 | +CH6O | 929.5295 (+4.1) | 13.5 | 7.6 | O-methylated BTX-2 |
| 10a | C50H74O16 | +H4O2 | 931.5090 (+4.3) | 13.5 | 3.7 | |
| 10b | C50H74O16 | +H4O2 | 931.5091 (+4.5) | 13.5 | 4.5 | |
| 11a | C51H74O16 | +CH4O2 | 943.5083 (+3.5) | 14.5 | 6.5 | |
| 11b | C51H74O16 | +CH4O2 | 943.5081 (+3.3) | 14.5 | 7.6 | |
| 11c | C51H74O16 | +CH4O2 | 943.5082 (+3.4) | 14.5 | 8.1 | |
| 12 | C60H87N3 O20S | +C10H17N3O6S | 1202.5654 (−1.9) | 18.5 | 4.2 | BTX-2-GSH |
| 13 | C60H89N3 O20S | +C10H19N3O6S | 1204.5820 (−1.1) | 17.5 | 3.5 | BTX-3-GSH |
Note that the RT of standards in UHPLC-HRMS(/MS) standard characterizations (Fig. S5) and those observed in incubation samples are shifted slightly due to instrumental variability across analyses as well as potential effects of the different matrices of incubation samples and standard solutions.
Identities confirmed by comparison with BTX-2, BTX-3, BTX-B5 reference standards and corresponding A-ring hydrolyzed standards.
Metabolites 5d and 5e likely represent a pair of diastereoisomers based on nearly identical RT and HRMS/MS spectra.