Identification of dihydroxyvitamin D3 metabolites by HPLC-UV, periodate cleavage, and LC-MS/MS analysis
After incubation of 25OHD3 with recombinant CYP3A4 or HLM, the vitamin D3 metabolites were extracted by LLE and reconstituted in mobile phase for HPLC-UV analysis. The fractions corresponding to eight peaks were collected, dried, and subjected to PTAD derivatization. The derivatives were then divided into two parts, one for LC-MS/MS analysis directly and the other for periodate cleavage followed by LC-MS/MS analysis. The results were compared with those for authentic standards, which were conducted under the same conditions. HPLC analysis was performed using method I and LC-MS/MS analysis was performed using method II. After reaction with NaIO4 or water as vehicle, metabolites were extracted by ethyl acetate; after evaporation, samples were reconstituted in acetonitrile (40%) for LC-MS/MS analysis. Experimental details are described under Materials and Methods.
| Vitamin D Metabolites | HPLC-UV RT | LC-MS/MS after PTAD Derivatizationa | Periodate Cleavage (Vicinal–OH Groups?) | Assigned Structures | |
|---|---|---|---|---|---|
| MRM (m/z) | RT | ||||
| min | min | ||||
| Peak 1 | 27.36 | 574 → 298 | 8.61; 10.18 | Yes | 24S,25(OH)2D3 |
| Peak 2 | 27.81 | 574 → 298 | 8.56; 10.13 | Yes | 24R,25(OH)2D3 |
| Peak 3 | 28.61 | 574 → 298 | 8.59; 9.97 | No | 23S,25(OH)2D3 |
| Peak 4 | 31.95 | 574 → 298 | 8.78; 9.81 | Yes | 25,26(OH)2D3 |
| Peak 5 | 32.62 | 574 → 314 | 11.31; 12.15 | Yes | 4β,25(OH)2D3 (M1) |
| Peak 6 | 34.01 | 574 → 314 | 11.15; 11.91 | Yes | 4α,25(OH)2D3 (M2) |
| Peak 7 | 34.70 | 574 → 298 | 11.37 | No | 23R,25(OH)2D3 |
| 574 → 314 | 11.62 | No | 1α,25(OH)2D3 | ||
| 574 → 314 | 12.36 | No | 1α,25(OH)2-3-epi-D3 | ||
| Peak 8 | 35.21 | 574 → 314 | 12.37 | No | 1α,25(OH)2-3-epi-D3 |
| 574 → 314 | 11.62 | No | 1α,25(OH)2D3 | ||
RT, retention time.