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SHORTCUT to CYP Enzyme Activity Monitoring

Nov 1, 2014 10:41:00 AM / by Dr. Feng Li

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.

LC-MS/MS cyp enzyme activity monitoring

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+


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



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

Topics: lc-ms/ms

Dr. Feng Li

Written by Dr. Feng Li

Feng (Frank) Li, Ph.D., is President of Alliance Pharma; he obtained his Ph.D. degree in Bioanalytical Chemistry jointly from Concordia University and the National Institute of Scientific Research (Canadian Doping Control Center) in Montreal, Canada. Subsequently, Dr. Li did his post-doctoral fellowship at the Biomedical Mass Spectrometry Facility at the Mayo Clinic in Rochester, Minnesota. Furthermore, Dr. Li has an M.Sc. degree in Natural Product Chemistry and a B.S. in Pharmacy. He has held leadership roles in the Department of Drug Discovery Metabolism at Phoenix International Life Sciences, Inc., a major CRO at the time in Montreal, Canada, which was later acquired by MDS Pharma Services; in the Drug Analysis group in the Department of Drug Metabolism and Pharmacokinetics (DMPK) at GlaxoSmithKline; and in the Drug Metabolism group in the Department of Drug Safety and Disposition at Cephalon, Inc. Dr. Li has extensive DMPK experience in discovery and developmental phases of drug development. With more than 20 years in the pharmaceutical biotechnology, and CRO industry, he is well versed in bioanalytical techniques for both qualitative (drug metabolite identification) and quantitative (PK/TK) drug analysis and has published numerous articles in the area of drug metabolite identification and quantitation.