High Sensitivity Analysis of Testosterone in Human Serum Using LCMS™-8060NX

Importance of the Topic

Testosterone is a critical steroid hormone used as a biomarker for endocrine disorders such as Cushing’s syndrome and hormone‐related tumors. Accurate and highly sensitive quantification of testosterone in human serum is essential for reliable clinical diagnostics and research in endocrinology.

Objectives and Study Overview

This application note demonstrates a method for ultra‐sensitive quantification of testosterone in human serum using the Shimadzu LCMS‐8060NX triple quadrupole mass spectrometer. The goal was to achieve reliable detection from 0.05 to 1000 pg/mL with high selectivity and accuracy.

Methodology and Instrumentation Used

The workflow combined protein‐free sample preparation with LC-MS/MS analysis under the following conditions:

• Sample Preparation
  • Standard solutions diluted in 50% methanol over 0.05–1000 pg/mL
  • Serum spiked to 1 pg/mL using steroid‐free human serum
  • Liquid–liquid extraction with hexane/ethyl acetate (9:1), centrifugation, evaporation, reconstitution in 50% methanol
• LC Conditions
  • System: Nexera X3
  • Column: Shim-pack Scepter C18 50×2.1 mm, 1.9 µm
  • Mobile phase A: 0.05 mM NH4F in water; B: methanol; gradient elution at 0.3 mL/min; injection 25 µL
• MS Conditions
  • Instrument: LCMS-8060NX, ESI positive mode, MRM
  • Transition: Testosterone m/z 289.25 → 97.15; internal standard [13C3] m/z 292.25 → 100.20
  • Interface voltage 1 kV, DL temp. 250 °C, block heater 500 °C, nebulizer gas 3 L/min

Main Results and Discussion

The calibration curve exhibited excellent linearity over 0.05–1000 pg/mL (R2 = 0.9996). At the lower limit of quantification (0.05 pg/mL), precision (%RSD) was 2.47% and accuracy ranged from 94.6% to 111.0%. Analysis of serum samples spiked at 1 pg/mL yielded a mean recovery of 105%, demonstrating minimal matrix interference and high method robustness.

Benefits and Practical Applications

This method allows quantification of testosterone at sub‐picogram levels with high selectivity, reducing cross‐reactivity issues common in immunoassays. It is well suited for clinical research, endocrinology studies, and therapeutic drug monitoring requiring trace‐level detection.

Future Trends and Possibilities of Use

Integration of this high‐sensitivity approach into routine clinical workflows could enable earlier detection of hormone dysregulation. Further development may include multiplexed steroid panels, automation of sample preparation, and coupling with microflow LC for reduced solvent consumption.

Conclusion

The LCMS-8060NX–based protocol delivers reliable, highly sensitive quantification of testosterone in human serum over a broad dynamic range. The combination of simple sample preparation, robust chromatography, and selective MRM detection ensures precise and accurate results suitable for advanced clinical and research applications.