Rapid and sensitive measurement of cortisol and 6-Hydroxycortisol in human urine using LC-MS/MS.
Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP metabolizing
enzyme. Cortisol can be converted to 6ß-hydroxycortisol (6ß-HC) by CYP 3A4. The latter is excreted in urine. Cortisol and 6ß-HC ratio in urine may act as CYP3A4 activity indicator.
Sample Preparation
- Human urine samples are spiked with internal standard and then extracted by liquid-liquid extraction with MTBE.
- Partial supernatant is dried, reconstituted, and injected into LC-MS/MS.
- STDs, LLOQ, and LQC are prepared in urine surrogate, PBS solution.
LC-MS/MS Method
Column: Aquity BEH C18, 50 X 2.1 mm, 1.7 μm
Mobile Phase: 0.1% formic acid in water and ACN
Flow Rate: 0.5 mL/min
Mass Spec: Sciex API 5500, ESI+
Results
| Analyte | Cortisol | 6ß-HC |
| Calibration range(ng/mL) | 0.4-200 | 2-1000 |
| Inter-Assay precision(%) | 2.4-7.3 | 1.9-7.4 |
| Inter-Assay accuracy(%) | 95.1-111.3 | 90.6-114.1 |
| Intra-Assay precision(%) | 0.7-1.2 | 1.7-4.3 |
| Intra-Assay accuracy(%) | 95.7-110.3 | 92.4-110.6 |
| Processed stability | 4 days | 4 days |
| Ben-Top stability | 18 hours | 18 hours |
| Freeze/Thaw stability | 4 cycles | 4 cycles |
| Long-term stability | 1 month | 1 month |
Conclusion
A rapid and sensitive LC-MS/MS method was validated for cortisol and 6ß-HC analysis
in human urine. Method accuracy, precision, repeatability, selectivity, F/T stability, processed sample stability, bench-top stability, and long term stability have been confirmed.
Scientists
Guodong (Gordon) Gu, Yifan Shi, and Yinghe Li
