Analytical determination of testosterone in human serum using an Agilent Ultivo Triple Quadrupole LC/MS

Significance of the Topic

This application note addresses the quantitation of free testosterone in human serum, a critical analyte in clinical research and endocrinology. Accurate measurement of endogenous hormones like testosterone is essential for diagnostic assessment, therapeutic monitoring, and biomedical research. Miniaturized yet robust LC/MS platforms can streamline laboratory workflows and improve access to high-performance analysis in routine settings.

Study Objectives and Overview

The study aims to evaluate the performance of the Agilent Ultivo Triple Quadrupole LC/MS in quantifying free testosterone in serum. Key goals include determining linearity, sensitivity, precision, and throughput in a fast six-minute LC/MS cycle. The investigation benchmarks the compact Ultivo system against larger conventional instruments to confirm comparable analytical capabilities.

Methodology and Instruments Used

Sample Preparation:

• Human serum was matrix-matched and spiked to create an 11-point calibration series (0.001–100 ng/mL).
• Protein precipitation: 250 µL serum mixed with 500 µL acetonitrile, vortexed, centrifuged, and diluted 1:1 with water.
• Internal standard (testosterone-d3) added to achieve a constant 25 ng/mL across samples.

Chromatography:

• Agilent 1290 Infinity II UHPLC system with Poroshell 120 EC-C18 column (2.1×50 mm, 2.7 µm).
• Mobile phases: 0.1% formic acid/5 mM ammonium acetate in water (A) and methanol (B).
• Gradient: 60% B at 0 min to 95% B at 4 min, hold to 5 min, return to 60% B at 5.1 min; total run 6 min.
• Flow rate 0.5 mL/min; column at 55 °C; injection 19 µL plus 1 µL internal standard; autosampler at 4 °C.

Mass Spectrometry:

• Agilent Ultivo Triple Quadrupole MS in positive MRM mode.
• Source: drying gas 300 °C/8 L/min, sheath gas 380 °C/12 L/min, nebulizer 50 psi, capillary +3000 V.
• Dodecapole Vortex collision cell, VacShield, Cyclone Ion Guide, Hyperbolic Quads for enhanced robustness.
• MRM transitions: testosterone m/z 289.2→109.1 (CE 18 V) and 289.2→97.0 (CE 17 V); testosterone-d3 m/z 292.2→97.0 (CE 17 V).

Main Results and Discussion

The calibration curve exhibited excellent linearity (1 pg/mL–100 ng/mL) with R2>0.999 and <5% RSD across three batches. Overlaid MRM chromatograms showed clear separation of analyte and internal standard at ~1.65 min. Analytical sensitivity allowed detection of 1 pg/mL testosterone against endogenous blank levels, leveraging a blank-offset feature to correct for background. Method precision and accuracy were maintained over the entire range in a rapid 6-minute cycle time.

Benefits and Practical Applications

This workflow offers:

• High-throughput analysis for clinical and research laboratories.
• Minimal maintenance due to compact design and advanced ion handling.
• User-friendly MassHunter software for streamlined acquisition and reporting.
• Reliable quantitation of low-level endogenous hormones in complex biological matrices.

Future Trends and Opportunities

Further studies should explore:

• Interference assessment across diverse serum and whole blood sources.
• Alternative sample preparation techniques (e.g., solid-phase extraction) for improved cleanup.
• Extension to other steroid hormones and multiplex panels.
• Integration with high-resolution MS for structural confirmation.

Conclusion

The Agilent Ultivo Triple Quadrupole LC/MS demonstrates robust, sensitive, and precise quantitation of free testosterone in human serum with a fast six-minute analysis time. The miniature instrument delivers performance comparable to larger systems, offering a practical solution for routine clinical and research laboratories.

Reference

1. Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray tandem MS. Analytical Biochemistry. 2010;402(2):121–128.