Simultaneous measurement of total testosterone and estradiol by liquid chromatography tandem mass spectrometry

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 358-380-Steroid Hormone Biosynthesis & Metabolism
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-369
Yuesong Wang*, Matthew Gatcombe, Jacob Farris, Julianne Cook Botelho and Hubert W Vesper
Centers for Disease Control and Prevention, Atlanta, GA
Effective diagnosis, treatment and prevention of hormone related diseases highly depend on accurate and comparable laboratory measurements. The CDC hormone standardization program is working with clinical, research, and public health laboratories and professional and medical organizations to improve the quality and comparability in hormone measurement. As part of this program, CDC is creating data on hormone levels in the U.S. population using the National Health and Nutrition examination Survey (NHANES). For this purpose, highly accurate analytical methods with appropriate sample throughput and specimen requirements are needed.

Isotope dilution (ID) liquid chromatography tandem mass spectrometry (LC-MS/MS) can provide the specificity, sensitivity, accuracy and throughput needed to perform measurements in large population surveys. In addition, LC-MS/MS enables the simultaneous measurement of steroid hormones.

Total testosterone (TT) and estradiol (E2) are released from serum (200 µl) binding proteins under acidic buffer conditions and isolated from proteins, phospholipids, and other serum matrix components through liquid-liquid extractions. All extraction procedures are performed with an automated 96-well plate platform to allow for high throughput. The LC separation is performed on a reverse phase LC column with a gradient of water and methanol and an ionization modifier. A triple quadrupole instrument is operated in electrospray positive-negative switching selected reaction monitoring (SRM) mode. E2 and its internal standard are quantified in negative-ion mode with the ion transition m/z 271 to 145 and 274 to 148, respectively. Simultaneously TT and its internal standard are quantified in positive-ion mode with transition m/z 289 to 97 and 292 to 100. In total, 32 steroid analogs were tested with no interference to this method. The calibration curve is 0.2-1000 pg/ml for estradiol and 0.2-1000 ng/dL for testosterone. The method can be used in serum from male and female including pediatrics, old men, and postmenopausal women.

Nothing to Disclose: YW, MG, JF, JCB, HWV

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