Quantitative analysis of estradiol and testosterone in plasma for clinical research using the TSQ Altis triple quadrupole mass spectrometer

Importance of the topic

Accurate measurement of sex steroids such as estradiol and testosterone in plasma is critical for clinical research into hormone-related disorders, endocrine therapies and physiological studies. Endogenous levels of these analytes are often in the low picogram per milliliter range, demanding highly sensitive and selective analytical methods. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) provides the necessary specificity and sensitivity for reliable quantitation in complex biological matrices.

Study objectives and overview

The primary goal was to establish a robust LC-MS/MS workflow on the Thermo Scientific TSQ Altis triple quadrupole mass spectrometer to quantitate estradiol and testosterone in human plasma. Key objectives included achieving low limits of quantitation (LLOQs), ensuring linearity across relevant concentration ranges, and demonstrating acceptable precision and accuracy for routine clinical research applications.

Methodology and used instrumentation

Sample preparation involved liquid-liquid extraction (LLE) of 400 µL plasma spiked with calibration standards (0.5–10 ng/mL) and isotope-labeled internal standards (testosterone-13C3 and estradiol-d5). Extraction was performed with methyl tert-butyl ether (MTBE), followed by evaporation and reconstitution in 50:50 methanol/water. Triplicate 10 µL injections were analyzed by LC-MS/MS.

Used instrumentation:

• Thermo Scientific Vanquish Flex Binary HPLC system
• Accucore aQ C18 Polar Endcapped column (100 × 2.1 mm, 2.6 µm)
• Mobile phase A: 0.5 mM ammonium fluoride in water; mobile phase B: methanol; column at 40 °C, 9-min gradient
• Thermo Scientific TSQ Altis triple quadrupole MS with heated electrospray ionization (HESI)
• Timed selected reaction monitoring (t-SRM) with cycle time of 0.4 s, monitored quantifier and qualifier transitions for each analyte and internal standard
• Data acquisition and processing via Thermo Scientific TraceFinder software

Main results and discussion

Limits of quantitation were defined by back-calculated accuracy within ±20%, ion ratio tolerance within 20%, and replicate precision ≤20% RSD. The method achieved:

• Testosterone LLOQ: 2 pg/mL (linearity 2–1,000 pg/mL); precision ≤8%, accuracy within ±19%
• Estradiol LLOQ: 20 pg/mL (linearity 20–10,000 pg/mL); precision ≤7%, accuracy within ±19%

Chromatographic separation delivered retention times around 4.8–4.9 minutes with clear resolution. The TSQ Altis instrument provided the sensitivity required for low-level detection in plasma.

Benefits and practical applications

The developed method offers:

• Superior sensitivity for ultra-low level quantitation in clinical studies
• High selectivity via SRM transitions to minimize matrix interferences
• Efficient sample throughput with a 9-minute run time
• Reliable performance suitable for pharmacokinetic, endocrinology and biomarker research

Future trends and potential applications

Advancements may include microflow LC to reduce solvent use and sample volume, further enhancement of MS sensitivity, higher-throughput multiplexed assays for broader steroid panels, and integration with automated sample preparation. Emerging applications could extend to personalized medicine, large-scale epidemiological studies and improved diagnostic testing in clinical laboratories.

Conclusion

The LC-MS/MS method on the TSQ Altis platform achieves low picogram-level quantitation of estradiol and testosterone in plasma with excellent precision, accuracy and selectivity. Its performance supports diverse clinical research needs and establishes a foundation for future enhancements in steroid analysis.

References

1. Xie X, Van Natta K, Wijeratne N, Martins C. Quantitative analysis of estradiol and testosterone in plasma for clinical research using the TSQ Altis triple quadrupole mass spectrometer. Thermo Fisher Scientific Technical Note 64973; 2017.