| Chart 3 Determination of 6β-Hydroxycortisol and cortisol in urine | |
| Urine | |
| Centrifuge at 1000g for 30min | |
| Supernatant | 1ml |
| 5μmol/l Fludrocortisone(F-E;I.S.) | 200μl |
| Ethyl acetate | 6ml |
| Vortex-mix for ca.3min |
|
| Organic layer | |
| Wash with 1mol/l NaOH(1 ml)and ultra pure water (2ml) Centrifuge at 1000g for ca. 3min
|
|
| Supernatant (Organic layer) | 4.5ml |
| Evaporate to dryness under N2 gas | |
| MeOH | 100μl |
| 39 mM CH3COOCu(II) | 20μl |
| Stand at room temperature for 1 h | |
| 1mg/ml DMB solution (50μl/ml β-mercaptoethanol, 50mg/ml Na2S2O4) | 100μl |
| Warm at 60℃ for 40min | |
| Apply onto HPLC(10μl)
|
|
| HPLC conditions | |
| Column:L-Column ODS(250X4.6mmi.d.;5μm) | |
| Mobile phase:MeOH-CH3CN-0.5M CH3COONH4(gradient elution) | |
| Flow-rate:1.0ml/min | |
| Fluorescence detection:Ex.350nm;Em.390nm | |
