Evaluation of Prelude SPLC and TSQ Endura Mass Spectrometer in Research Analysis of Testosterone in Human Serum

Importance of the Topic

Accurate measurement of testosterone in human serum underpins clinical research into endocrine disorders, hormone replacement therapies and sports medicine. High sensitivity, reproducibility and throughput are essential for reliable data in large-scale studies and routine laboratory operations.

Study Objectives and Overview

This study compares two workflows for testosterone quantitation by coupling a Thermo Scientific Prelude SPLC system to a TSQ Endura triple quadrupole mass spectrometer. One workflow employs online cleanup via TurboFlow technology, while the other uses offline liquid–liquid extraction. Both approaches share mass spectrometer settings and aim to assess performance metrics such as limit of quantitation, precision, accuracy, matrix effects and sample throughput.

Methodology

The online method uses a TurboFlow column for automated extraction, followed by separation on an Accucore aQ analytical column. Serum samples (200 μL) are diluted with water/methanol containing an internal standard, vortexed, centrifuged and injected. The offline method relies on methyl-tert-butyl ether liquid–liquid extraction, concentration of extracts and injection of 50 μL onto the same analytical column. Both methods utilize gradient mobile phases with ammonium formate and formic acid modifiers.

Used Instrumentation

• Thermo Scientific Prelude SPLC system with TurboFlow online extraction.
• Thermo Scientific TSQ Endura triple quadrupole mass spectrometer with heated electrospray ionization (HESI).
• Thermo Scientific Cyclone-P TurboFlow column (50 × 0.5 mm).
• Thermo Scientific Accucore aQ analytical column (50 × 3.0 mm, 2.6 μm).

Main Results and Discussion

Both workflows achieve low limits of quantitation (20 pg/mL online, 10 pg/mL offline) and broad calibration ranges up to 10,000 pg/mL. Intra- and inter-assay precision (%RSD) remained below 11% at low quality control levels and below 6% at high levels. Accuracy across calibration curves stayed within ±8%. Matrix effects were minimal, and dilution integrity was confirmed for up to 16-fold dilution, with recoveries near 100%. Carryover remained below 20% of the lower limit signal.

Benefits and Practical Applications

• Online cleanup reduces manual handling, lowers solvent consumption and shortens run time to 4 minutes per sample.
• Offline extraction provides flexibility for laboratories without online SPE but requires longer sample preparation and a 6.5 minute analysis.
• Both methods deliver robust performance suitable for clinical research, QC assays and high-throughput hormone monitoring.

Future Trends and Potential Applications

Advances in multiplexed SPLC configurations and faster scanning mass analyzers may further double throughput and enable simultaneous quantitation of multiple steroids. Integration with laboratory information management systems (LIMS) and automated data processing pipelines will streamline workflows and support personalized medicine studies.

Conclusion

The Prelude SPLC–TSQ Endura platform offers two validated workflows for testosterone analysis in human serum, combining sensitivity, precision and speed. The online TurboFlow approach excels in automation and efficiency, while the offline method remains a viable alternative for laboratories preferring conventional extraction. Both workflows meet stringent performance criteria for clinical research applications.