An LC-MS Clinical Research Method for Measuring Male Androgens in Serum

Importance of the Topic

Clinical measurement of male androgens plays a crucial role in assessing hormonal balance and diagnosing endocrine disorders. While testosterone remains the primary marker in most clinical labs, expanding the panel to include androstenedione, dihydrotestosterone and dehydroepiandrosterone offers a more comprehensive androgen profile and supports research into hypogonadism, prostate disease and metabolic conditions.

Aims and Study Overview

This study aimed to develop a rapid LC MS based clinical research method capable of quantifying four key androgens from minimal serum volume. The approach combines online solid phase extraction for sample cleanup with tandem mass spectrometry to achieve simultaneous measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone within a single injection.

Methodology and Instrumentation

Sample Preparation and Online SPE

• 100 microliters of serum mixed with zinc sulfate and acetonitrile containing deuterated internal standards
• Centrifugation followed by automated extraction via online SPE using a MassTrak C18 cartridge
• SPE conditioning with methanol and water and direct elution into the LC system

LC MS MS Analysis

• UPLC separation on a 2.1 x 50 mm HSS SB C18 column
• Gradient elution from 50 to 70 percent methanol with 0.05 percent formic acid over three minutes
• Flow rate of 0.45 milliliters per minute
• Detection with a tandem quadrupole mass spectrometer in positive ion mode
• Optimized quantifier and qualifier transitions for each androgen

Main Results and Discussion

Chromatographic separation of all four analytes was achieved within a 6.5 minute runtime. Calibration curves were linear up to 50 nanomoles per liter for testosterone, androstenedione and dehydroepiandrosterone and up to 5 nanomoles per liter for dihydrotestosterone with correlation coefficients above 0.99. Lower limits of quantitation were in the low nanomolar range. Recovery studies showed mean extraction efficiencies between 91 and 101 percent. Post column infusion experiments indicated minimal ion suppression. Comparison with established single analyte methods yielded regression slopes near unity for testosterone and androstenedione.

Benefits and Practical Applications

• Simultaneous multiplex analysis of four androgens in a single run
• Minimal sample volume requirement of 100 microliters
• Short analysis time without complex derivatization
• High sensitivity and robust quantitation suitable for clinical research

Future Trends and Opportunities

Advances in online sample preparation and chromatography may expand multiplex panels to additional steroid hormones and metabolites. Integration with high resolution mass spectrometry could further improve specificity and facilitate targeted metabolomics. Automation and miniaturization of sample handling will enhance throughput for large clinical studies and support personalized medicine approaches.

Conclusion

The presented online SPE LC MS MS method delivers a fast and efficient platform for measuring a panel of male androgens in serum. It combines high analytical sensitivity with streamlined sample preparation to support high throughput clinical research without complex derivatization or extended chromatography.

Reference

Owen L Keevil B An LC MS Clinical Research Method for Measuring Male Androgens in Serum Waters Corporation 2014