Endocrine Abstracts

Introduction: The measurement of male androgens in most NHS laboratories is often limited to testosterone alone. To more accurately determine the androgen status in men the measurement of other androgens such as DHT and DHEA would be beneficial however these are difficult to measure without derivatisation. We report a combined LC–MS/MS assay for the measurement of testosterone, androstenendione, DHT and DHEA on a small sample volume.

Methods: Zinc sulphate (100 μl) was added to 100 μl of sample. After mixing, acetonitrile containing internal standards was added and was further mixed. The samples were centrifuged before analysis. Samples were extracted using an automated on-line solid phase extraction on a C18 cartridge by a Waters Acquity/OSM and analytes were measure using a Waters TQS tandem mass spectrometer.

Results: Chromatographic separation was achieved between all four androgens. The run time was 6.2 min per sample. The lower limit of quantitation was 0.2 nmol/l for testosterone, 0.3 nmol/l for androstenedione, 0.17 nmol/l for DHT and 2 nmol/l for DHEA. The CV of the assay for testosterone and DHT was <6%, androstenedione was <9% and DHEA was <5%. The testosterone and androstenedione gave the following comparisions with the routine LC–MS/MS assay: Testosterone (combined)=1.01×existing assay+0.07 nmol/l and androstenedione (combined)=1.09×existing assay−0.29 nmol/l.

Discussion: We have developed a rapid assay for the LC–MS/MS measurement of testosterone, androstenedione, DHT and DHEA in a routine clinical laboratory. The assay requires a small volume of sample and all analytes are measured simultaneously without derivitisation achieved by using a high sensitivity mass spectrometer. The assay is rapid and simple to prepare and has the potential for routine clinical use or for the analysis of large numbers of samples involved in clinical trials.