Analysis of Testosterones Using a Core Enhanced Technology Accucore HPLC Column

Importance of the Topic

The accurate and rapid analysis of testosterone and related steroid metabolites is critical for anti-doping control, clinical diagnostics and pharmaceutical quality assurance. Monitoring compounds such as 11-ketotestosterone, epitestosterone and 19-nortestosterone helps enforce regulatory compliance in sports and supports therapeutic drug monitoring in medical settings.

Objectives and Study Overview

This application note evaluates the performance of a superficially porous Thermo Scientific Accucore RP-MS HPLC column for the fast separation and quantification of three testosterone analytes. Major aims include achieving efficient resolution within minimal run times, demonstrating method precision and highlighting suitability for high-throughput workflows.

Methodology

Sample Preparation

• A working standard of each testosterone analogue (11-ketotestosterone, epitestosterone, 19-nortestosterone) at 50 µg/mL in the mobile phase.

Chromatographic Conditions

• Column: Accucore RP-MS, 2.6 µm, 100 × 2.1 mm
• Mobile phase: 60:40 (v/v) water/acetonitrile
• Flow rate: 0.6 mL/min
• Column temperature: 40 °C
• Injection volume: 1 µL
• Detection: UV at 254 nm
• Measured backpressure: 300 bar

Used Instrumentation

• Thermo Scientific Accucore RP-MS HPLC column (2.6 µm, 100 × 2.1 mm)
• Thermo Scientific Accela HPLC system
• Fisher Scientific HPLC-grade water and acetonitrile
• NSC Mass Spec certified 2 mL clear vials with bonded PTFE silicone caps

Main Results and Discussion

The three steroid analytes were fully resolved in under two minutes, exhibiting narrow, symmetrical peaks (asymmetry 1.11–1.48). Precision data from six replicates showed retention time %RSD ≤ 0.06% and peak area %RSD ≤ 0.44%. The core-shell particle design minimized secondary interactions and maintained low system backpressure, enabling high chromatographic efficiency.

Benefits and Practical Applications

• Exceptional throughput: analysis completed in under two minutes supports large sample batches.
• Low backpressure: compatible with conventional HPLC systems, avoiding specialized high-pressure hardware.
• Superior peak shape: enhances quantification accuracy, even for trace-level analytes.
• Robust reproducibility: ideal for routine anti-doping testing, clinical labs and pharmaceutical quality control.

Future Trends and Potential Applications

Emerging developments in superficially porous particle technology will further reduce analysis times and improve sensitivity, especially when coupled with tandem mass spectrometry for enhanced selectivity. Advances in microflow and nano-LC platforms, along with fiber-optic UV detection, may enable point-of-care steroid profiling in decentralized clinical or field settings.

Conclusion

The Accucore RP-MS column offers a powerful combination of speed, efficiency and reproducibility for testosterone metabolite analysis. Its low-pressure operation and robust performance make it a valuable asset for high-throughput laboratories across anti-doping, clinical diagnostics and pharmaceutical research.