HPLC radiochromatographic LC/MS-MS, and 600-MHz NMR characteristics for EP and its metabolites identified in human 0-48 hour urine and 0-96 hour feces
Compound | HPLC Radiochromatographic Retention Time | Molecular Weight | APCI MS-MS Spectral Ionsa | 600-MHz Proton NMR Chemical Shiftsb |
|---|---|---|---|---|
| min | m/z | ppm | ||
| 3β, 6β, 21-OHEP | 14.0 | 448 | 466(MNH4+, 78), 431 (100), 430(17), 413(10), 399(6), 398(9), 395(4), 381(9), 353(14), 345(5), 317(12), 219(6), 195(30), 177(8) | 5.57s(C-4); 4.38dd J 10 8.9 (C-21); 4.34d J 2.1 (C-6α); 4.03 ddd J 10.0, 6.4 2.0 (C-3α); 3.55s (-OCH3); 3.06d J 5.1 (C-11); 2.87dd J 11.0, 5.1 (C-8); 2.72dd J 4.9, 2.1 (C-7); 2.66dd J 13.7 8.9 (C-20β); 1.66d J 14.3 (C-12α); 1.60dd J 14.0, 5.4 (C-12β); 1.47s (C-19 CH3); 0.901s (C-18 CH3) |
| 2α, 3β, 6β-OHEP | 15.4 | 448 | 466(MNH4+, 66), 465(22), 449(6), 431(78), 430(8), 413(52), 399(7), 395(11), 381(15), 363(8), 353(11), 335(10), 317(7), 229(7), 221(34), 203(15), 193(100), 175(12), 161(23), 121(5) | 5.56d J 2.1 (C-4); 4.45d J 2.6 (C-6α); 3.74dd J 6.5, 2.2 (C-3); 3.54s (-OCH3); 3.42ddd J 9.7, 6.51 4.1 (C-2β); 3.14 d J 5.4 (C-11); 2.86dd J 11.0, 5.0 (C-8); 2.74dd J 4.9, 2.3 (C-7); 1.79d J 15.3 (C-12α); 1.65dd J 14.6, 5.5 (C-12β); 1.59dd J13.9, 4.1 (C-1β); 1.01dd J 13.9, 9.7 (C-1α); 1.45s (C-19 CH3); 0.936s (C-18 CH3) |
| 3α, 6β, 21-OHEP | 16.2 | 448 | 466(MNH4+, 46), 431(100), 413(11), 399(14), 381(10), 353(7), 325(6), 237(6), 195(39), 171(5) | 5.70d J 4.5 (C-4); 4.39dd J 10.0 8.9 (C-21); 4.35d J 2.0(C-6α); 3.97td J 2×4.5, 1.5 (C-3β); 3.56s (-OCH3); 3.06d J 5.3 (C-11); 2.88dd J 11.0, 4.9 (C-8); 2.73dd J 4.9, 2.2 (C-7); 2.67dd J 13.5 8.7 (C-20β); 1.68d J 14.6 (C-12α); 1.62dd J 14.4, 5.4 (C-12β); 1.369s (C-19 CH3); 0.907 (C-18-CH3); 0.874dt J 12.8, 2×3.6 (C-1β) |
| 6β, 15α-OHEP | 16.5 | 446 | 447(MH+, 100), 430(25), 429(21), 415(35), 397(22), 387(10), 379(11), 351(20), 333(10), 219(35), 201(10), 193(15), 179(10), 165(8), 119(10) | 5.84s(C-4); 4.56d J 2.3 (C-6α); 4.1ddd J 9.6, 7.4, 2.1 (C-15β); 3.59s (-OCH3); 3.18dd J 11.7, 4.6 (C-8); 3.15d J 5.5 (C-11); 3.14dd J 4.6, 2.4 (C-7); 1.87d J 14.4 (C-12α); 1.65dd J 14.7, 5.5 (C-12β); 1.59s (C-19 CH3); 1.38ddd J 13.2, 5.2, 2.4 (C-1β); 0.980d J 1.0(C-18 CH3) |
| 6β, 21-OHEPc | 16.9 | 446 | 447(MH+, 100), 446(11), 428(5), 415(11), 397(28), 369(45), 351(5), 343(5), 341(7), 340(9), 325(5), 279(7), 211(14) | 5.82s(C-4); 4.48d J 2.3 (C-6α); 3.57s (-OCH3); 3.10d J 5.2 (C-11); 2.95dd J 11.3, 4.6 (C-8); 2.82dd J 4.5, 2.3 (C-7); 2.71dd J 13.3, 8.4 (C-20β isomer B); 2.67dd J 13.6, 8.7 (C-20β isomer A); 1.72d J 14.7 (C-12α isomer B); 1.71d J 14.1 (C-12α isomer A); 1.65dd J 14.4, 5.3 (C-12βisomers A and B); 1.57s (C-19 CH3); 1.37 ddd J 13.4, 5.4, 2.3(C-1β); 0.928s (C-18 CH3 isomer A); 0.903s (C-18 CH3 isomer B) |
| 3β, 6β-OHEP | 18.9 | 432 | 450(MNH4+, 22), 449(6), 433(29), 415(100), 401(6), 397(66), 383(11), 379(20), 365(12), 355(6), 347(7), 337(25), 319(14), 213(18), 195(31), 163(10) | 5.58s (C-4); 4.34d J 2.1 (C-6α); 4.03ddd J7.9, 6.4, 1.9 (C-3β); 3.53s (-OCH3); 3.02d J 5.4 (C-11); 2.95dd J 11.3, 4.6 (C-8); 2.82dd J 4.6, 2.3 (C-7); 1.74d J 14.4 (C-12α); 1.62dd J 12.6, 5.5 (C-12β); 1.48s (C-19 CH3); 1.03ddd J 12.9, 4.2, 3.5 (C-1β); 0.949 (C-18 CH3) |
| 3α, 6β-OHEP | 19.8 | 432 | 450(MNH4+, 42), 449(26), 415(100), 414(6), 397(8), 383(20), 365(9), 347(6), 337(13), 221(7), 205(9), 195(40), 177(8), 163(5) | 5.70d J 4.5 (C-4); 4.35d J 2.2 (C-6α); 3.97m (C-3β); 3.54s(-OCH3); 3.08d J 5.4 (C-11); 2.89dd J 11.1, 4.6 (C-8); 2.73dd J 4.9, 2.2 (C-7); 1.75d J 14.8 (C-12α); 1.63dd J 14.6, 5.5 (C-12β); 1.38s (C-19 CH3); 0.953s (C-18 CH3); 0.878dt J 12.6, 2 × 3.5 (C-1β) |
| 6β-OHEP | 22.2 | 430 | 431(MH+, 100), 430(14), 413(8), 399(5), 398(9), 395(6), 381(23), 380(17), 363(26), 353(42), 345(5), 337(6), 335(10), 317(6), 293(5), 221(7), 211(16), 193(5), 179(20), 111(5) | 5.82s (C-4); 4.48d J 2.1 (C-6α); 3.55s (-OCH3); 3.12d J 5.3 (C-11); 2.95dd J 11.3, 4.6 (C-8); 2.82dd J 4.6, 2.3 (C-7); 1.79d J 14.7 (C-12α); 1.67dd J 14.7, 5.4 (C-12β); 1.57s (C-19 CH3); 1.38ddd J 13.2, 5.2, 2.4 (C-1β); 0.976d J 1.0 (C-18 CH3) |
| 21-OHEP | 23.5 | 430 | 431(MH+, 100), 430(7), 413(4), 399(6), 371(21), 353(14), 281(5), 237(7), 163(40), 147(6), 121(8) | 5.74 (C-4); 4.39dd J 10.1, 8.8 (C-21); 3.59s (-OCH3); 3.10d J 5.2 (C-11); 2.86td J2x5.2, 1.9 (C-7); 2.75ddd J15.6, 5.3, 2.2 (C-6β); 2.66dd J 13.6, 8.7 (C-20β); 2.61dd J 15.2, 1.9 (C-6α); 2.59dd J 11.1, 4.7 (C-8); 1.71d J 14.5 (C-12α); 1.64dd J 14.4, 5.3 (C-12β); 1.46s (C-19 CH3); 1.42ddd J 13.3, 5.2, 2.4 (C-1β); 0.976bs (C-18 CH3) |
| EP | 28.4 | 414 | 415(MH+, 100), 414(9), 397(9), 383(5), 379(5), 365(8), 355(21), 347(7), 337(25), 319(12), 221(5), 203(11), 195(5), 163(35), 121(6) | 5.75 (C-4); 3.56s (-OCH3); 3.12d J 5.3 (C-11); 2.86dt J 2.0, 2x5.2 (C-7); 2.76ddd J 15.6, 5.4 2.2 (C-6β); 2.63dd J 15.5, 2.0 (C-6α); 2.59dd J 11.2, 4.7 (C-8); 2.02ddd J 13.3, 4.8, 0.7 (C-1β); 1.78d J 14.8 (C-12α); 1.66dd J 14.7, 5.4 (C-12β); 1.48s (C-19 CH3); 1.42ddd J 13.3, 5.4, 2.4 (C-1β); 0.97d J 1.2 (C-18 CH3) |
↵ a The parent ion is indicated in each case as either the ammonium adduct, MNH4+, or the protonated species, MH+. Percentage relative abundance is in parentheses.
↵ b Chemical shifts are given in ppm referenced to the residual proton in deuterated acetonitrile (1.93 ppm), and coupling constants are given in Hz. The analyses are based on the assumption that the spectra are first order. The proton stereochemistry is indicated. dd = doublet of doublet; ddd = doublet of doublet of doublet; td = triplet of doublets; dt = doublet of triplets.
↵ c Mixture of both C-21 isomers; isomer A is the major isomer.