LC-MS/MS Method for the Determination of Testosterone using an Accucore C8 HPLC Column

Importance of the Topic

Monitoring testosterone levels plays a crucial role in assessing hormone replacement therapy efficacy and detecting illicit anabolic steroid use in sports. Rapid and reliable quantification supports clinical decision-making and ensures compliance with anti-doping regulations.

Aims and Study Overview

This study demonstrates a fast, sensitive LC-MS/MS method for testosterone determination using a Thermo Scientific Accucore C8 column. The goal was to achieve sub-2-minute cycle times while preserving peak shape and quantitative precision.

Methodology

Chromatographic separation was performed on a 2.6 µm, 50 × 2.1 mm Accucore C8 column with a water/0.1% formic acid to acetonitrile/0.1% formic acid gradient ramping from 5% to 95% organic in 0.8 minutes at 1.5 mL/min and 60 °C. A 5 µL injection volume and dual wash solvents minimized carryover.

Used Instrumentation

• HPLC System: Thermo Scientific Accela 600
• MS Detector: Thermo Scientific TSQ Vantage with HESI source
• Core Enhanced Technology column: Accucore C8, 2.6 µm
• Data Processing: Thermo Scientific LC QUAN software

Main Results and Discussion

Testosterone eluted at 0.73 minutes with excellent symmetry and minimal tailing. The method delivered a total run time of 1.5 minutes. Six replicate injections of a 20 ng/mL standard yielded retention time RSD of 0.22% and peak area RSD of 3.01%, demonstrating high precision.

Benefits and Practical Applications

Key advantages include high throughput, low backpressure compared to sub-2 µm phases, robust peak shape, and reproducible quantitation. The method suits clinical laboratories monitoring therapeutic levels and anti-doping analyses requiring rapid turnaround.

Future Trends and Opportunities

Advances may involve integrating high-resolution mass spectrometry for improved selectivity, automating sample preparation workflows, and extending the approach to multiplex steroid panels for comprehensive endocrine profiling.

Conclusion

The optimized LC-MS/MS protocol using an Accucore C8 column provides a fast, reliable tool for testosterone analysis, combining speed with analytical performance suitable for diverse regulated environments.