High sensitivity measurement of androgens by two-dimensional-high pressure liquid chromatography-tandem mass spectrometry

Dehydroepiandrosterone and androstenedione are precursors of sex hormones; testosterone is a bioactive precursor of steroids responsible for development of secondary male characteristics. Abnormal androgen production may cause changes in gender-specific physiology and measurements are useful in diagnosing androgen and estrogen related disorders. We developed a high sensitivity two-dimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measurement of dehydroepiandrosterone, androstenedione, and testosterone. Stable isotope-labeled internal standards were added to 200 ?L of serum and extracted into methyl-tert-butyl ether. The solvent was evaporated and steroids derivatized with hydroxylamine. Instrumental analysis was performed on an API 4000 (Applied Biosystems) LC-MS/MS utilizing a TurboIonsprayTM source in positive multiple reaction monitoring (MRM) mode. Quantitation was performed with AnalystTM software. The quantitative MRM transitions monitored were (m/z): 304 to 253 Da for DHEA; 306 to 255 Da for d2-DHEA; 317 to 124 Da for androstenedione; 324 to 128 Da for d7-androstenedione; 304 to 124 Da for testosterone; and 307 to 112 Da for d3-testosterone. Two-dimensional chromatographic separation was performed using a Phenomenex Gemini C6 cartridge as the first dimension and a Phenomenex Gemini C18 column as the second dimension. Injection-to-injection interval was 4.5 minutes. The assay was linear up to 9.2 ng/mL for DHEA, 42.5 ng/mL for androstenedione, and 25.8 ng/mL for testosterone.