Determination of Testosterone in Human Serum using Ultivo Triple Quad LC/MS

Importance of the Topic

Free testosterone is a critical biomarker in endocrinology and clinical research. Accurate measurement informs diagnosis and treatment of hormonal disorders, monitoring therapy efficacy, and quality control in pharmaceutical development.

Objectives and Overview

The study aimed to develop a fast, sensitive, and robust method to quantify free testosterone in human serum using the Agilent Ultivo triple quadrupole LC/MS system. It evaluated calibration linearity, sensitivity, precision, and robustness within a typical laboratory context.

Experimental Methodology and Instrumentation

• Sample Preparation: Protein precipitation of 250 µL serum with 500 µL acetonitrile, centrifugation, supernatant dilution and internal standard addition.
• Liquid Chromatography: Agilent 1290 Infinity UHPLC with Poroshell 120 EC-C18 (2.1×50 mm, 2.7 µm), 0.5 mL/min flow, gradient from 60% to 95% organic in 4 min, total cycle time 6 min.
• Mass Spectrometry: Agilent Ultivo QqQ in positive AJS mode; gas temp 300 °C, drying gas 8 L/min, nebulizer 50 psi, sheath gas 12 L/min at 380 °C, capillary voltage 3000 V, cell accelerate voltage 9 V; MRM transitions 289.2→109.1, 289.2→97.0 for testosterone and 292.2→97.0 for d3-labeled internal standard; Q1/Q3 resolution 0.7 unit.

Main Results and Discussion

The method achieved a lower limit of quantitation of 1 pg/mL (20 fg on-column) and maintained linearity up to 100 ng/mL with R²>0.999. Triplicate analysis across three batches yielded %RSD<5% at all calibration points. Blank serum showed minimal endogenous interference, while a 1 pg/mL spike produced clear, high signal-to-noise responses. The Quant-My-Way interface simplified data processing and method execution.

Benefits and Practical Applications

The compact Ultivo system offers:

• Reduced footprint and maintenance demands;
• Streamlined workflow for non-expert users;
• High throughput with 6-min analysis cycles;
• Robust performance suitable for clinical, research, and QA/QC laboratories.

Future Trends and Applications

Further studies will explore matrix effects across diverse serum and blood samples, refine sample preparation techniques, and expand the assay to additional steroid hormones. Automation and high-throughput adaptations may enhance screening capabilities.

Conclusion

The Agilent Ultivo triple quadrupole LC/MS method delivers excellent sensitivity, precision, and linearity for free testosterone quantitation in human serum, meeting the demands of routine clinical and research laboratories with rapid 6-minute cycle times.

Reference

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