Category: Other - Androgen biomarkers

Monitor: 24

24 - DEVELOPMENT AND VALIDATION OF A LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY BIOANALYTICAL METHOD FOR THE 11-OXYGENATED ANDROGENS 11-KETOTESTOSTERONE, 11-HYDROXYTESTOSTERONE AND 11-HYDROXYANDROSTENEDIONE IN SERUM OR PLASMA

Thursday, Apr 25
1:00 PM – 1:30 PM

Objective : We sought to develop a bioanalytical method for 11-oxygenated androgens for clinical use.


Methods : An analytical method was developed using a TX-4 HPLC system (Thermo-Fisher) with Agilent® 1200 pumps (Agilent Technologies, Inc.) and a Sciex® 5000 (Danaher) triple quadrupole mass spectrometer.  Independent calibration curves were prepared for each analyte in depleted serum (Golden West Biologicals).  Sample preparation consisted of isotope dilution using a cocktail of three deuterated heavy isotope internal standards (IsoSciences) followed by Liquid-Liquid Extraction.  A reversed phase C8 analytical column (3.0x50mm, 2.7 um) was used with a water/methanol/acetonitrile solvent gradient to achieve chromatographic separation of all isobars in under 4 minutes.  Positive mode atmospheric pressure chemical ionization (APCI) was used for detection in Multiple Reaction Monitoring (MRM) mode.


Results : Analytical sensitivity was between 1 – 3 ng/dL for each analyte and the analytical measurement range was up to 1,000 ng/dL (up to 10,000 ng/dL with dilution).  Inter-assay precision ranged from 4.6–9.5% for 11-ketotestosterone (11KT), 6.0–13.0% for 11-hydroxyandrostenedione (11OHA) and 6.0–10.0% for 11-hydroxytestosterone (11OHT).  Accuracy ranged from 100.7-106.4% for 11KT, 99.3-113% for 11OHA and 98.5-101% for 11OHT.  Reference intervals for both female and male adults were developed for 11KT (Female, 6-70 ng/dL; Male 9-74 ng/dL), 11OHA (Female, 23-338 ng/dL; Male, 41-311 ng/dL) and 11OHT (Female, <53 ng/dL; Male, 5-48 ng/dL).


Discussion : It has long been known that the adrenal gland is capable of metabolizing androstenedione to 11-beta androstenedione via the 11b-hydroxylase cytochrome P450 mixed function oxidase.  Recent studies have demonstrated that some 11-oxygenated androgen metabolites, which were previously believed to be insignificant, can be present at high concentrations and are biologically active with similar potencies as testosterone or dihydrotestosterone.  Elevated concentrations of 11-oxygenated androgens have been found in patients with classic 21-hydroxylase deficiency, polycystic ovary syndrome and castration resistant prostate cancer.  


Conclusion : A fully validated assay for 11-oxygenated Androgens is commercially available and may be useful in the investigation of castration resistant prostate cancer, 21-hydroxylase deficiency, and polycystic ovary syndrome.

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Bill Curtin

Laboratory Director
Endocrine Sciences, Laboratory Corporation of America® Holdings
Northridge, California

He received a BS in Biochemistry at the University of New Mexico where he developed a thin-layer chromatography method for separating deoxy-ribonucleotides from ribonucleotides, and an MS in Chemistry at California State University, Northridge where he developed a chemiluminescent immunoassay for the steroid hormone pregnenolone. He began working in steroid analysis (radioimmunoassay) in 1988 at Endocrine Sciences. He began working with mass spectrometry in 2003 and has been doing assay development for small molecules by mass spectrometry since 2010. He is licensed by the state of California as a clinical chemist and is the director of the mass spectrometry lab at Endocrine Sciences/Laboratory Corporation® of America.

Brett Holmquist

Associate Vice President
Endocrine Sciences, Laboratory Corporation of America® Holdings
Calabasas Hills, California

Dr Holmquist is board certified by the American Board of Clinical Chemistry, a fellow of the American Academy of Clinical Chemistry, licensed by the state of California as a clinical chemist, and certified as a New York State laboratory director. He earned his PhD in biochemistry and molecular biology from the University of California, Riverside. He has worked for more than 30 years in the life science industry, including more than 15 years in the diagnostic laboratory industry. He is a member of the Endocrine Society, the American Society for Mass Spectrometry, the American Society for Clinical Pathology, the American Society for Clinical Laboratory Science, and the American Association of Clinical Chemistry.

Walt Chandler

Technical Director
Endocrine Sciences, Laboratory Corporation of America® Holdings

Dr Chandler received his PhD in biochemistry from the University of California, Los Angeles. He has more than 40 years of experience in diagnostic testing of steroid hormones and has been published in numerous professional journals. Dr Chandler is a past recipient of the National Institutes of Health Cell and Molecular Biology National Research Service Award. He is licensed by the state of California as a clinical chemist and certified as a New York State laboratory director. He is a member of the Endocrine Society, the Pediatric Endocrine Society, and the American Association of Clinical Chemistry.