Table 2.
Conditions for the LC-MS/MS analyses
| HPLC | ESI-MS/MS conditions | ||||
|---|---|---|---|---|---|
| Chromatographic conditions | Electrospray conditions | ||||
| Column dimensions | 2.1 × 50 mm | Ionization mode | ESI (+) | ||
| Stationary phase | C-18 | Capillary voltage | 3500 V | ||
| Particle size | 3 μm | Source temperature | 150 °C | ||
| Column temperature | 50 °C | Desolvation temperature | 650 °C | ||
| Mobile phase A | ACN/H20 | Desolvation gas flow | 650 L/h | ||
| 5:95 + 0.1% FA | |||||
| Mobile phase B | ACN/H20 | ||||
| 95:5 + 0.1% FA | |||||
| Flow rate | 1 mL/min | ||||
| Injection volume | 5 μL | ||||
| Autosampler temperature | 5 °C | ||||
| Gradient | MS/MS conditions | ||||
| Time [min] | B% | Dwell time | 0.012 s | ||
| Initial | 20 | MS/MS | MRM | ||
| 5.00 | 100 | ||||
| 5.50 | 100 | Compound | Transition | CV | CE |
| 5.51 | 20 | Uroporphyrin | 831 > 727 | 10 | 112 |
| 6.00 | 20 | 831 > 623 | 10 | 112 | |
| 831 > 655 | 10 | 112 | |||
| 7-Carboxylporphyrin | 787 > 623 | 8 | 118 | ||
| 787 > 682 | 8 | 120 | |||
| 787 > 670 | 8 | 120 | |||
| 6-Carboxylporphyrin | 743 > 639 | 18 | 110 | ||
| 743 > 507 | 18 | 110 | |||
| 743 > 521 | 18 | 110 | |||
| 5-Carboxylporphyrin | 699 > 463 | 4 | 120 | ||
| 699 > 495 | 4 | 108 | |||
| 699 > 639 | 4 | 90 | |||
| Coproporphyrin | 655 > 522 | 10 | 120 | ||
| 655 > 536 | 10 | 120 | |||
| 655 > 595 | 10 | 102 | |||
| Hemin | 616 > 482 | 64 | 120 | ||
| 616 > 497 | 64 | 116 | |||
| 616 > 557 | 64 | 82 | |||
| Mesoporphyrin | 568 > 478 | 2 | 102 | ||
| 568 > 508 | 2 | 74 | |||
| 568 > 449 | 2 | 74 | |||
| Protoporphyrin | 563 > 444 | 20 | 110 | ||
| 563 > 489 | 20 | 96 | |||
| 563 > 504 | 20 | 78 | |||
CV cone voltage, CE collision energy