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.
- 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.
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+
|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|
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.
Guodong (Gordon) Gu, Yifan Shi, and Yinghe Li