Achieving high sensitivity and robustness – analysis of estrone and estradiol in human serum by TSQ Altis mass spectrometer for clinical research

Significance of the Topic

Estrogens like estrone and estradiol regulate key physiological processes but circulate at very low concentrations that challenge routine immunoassays. Sensitive and specific quantification at the low picogram per milliliter level in human serum is essential for advancing clinical research into steroid hormone biology.

Study Objectives and Overview

This work aimed to establish a robust, reproducible LC-MS/MS method using a Thermo Scientific TSQ Altis mass spectrometer and Vanquish Horizon UHPLC to quantify estrone and estradiol in human serum down to 2 pg/ml without any derivatization step.

Methodology

• Sample preparation by liquid–liquid extraction: 200 µl of serum or control matrix spiked with analytes and internal standards, extracted with tert-butyl methyl ether, evaporated, and reconstituted in 30% methanol.
• Chromatographic separation on a 50 × 2.1 mm, 2.6 µm Accucore Bi-Phenyl column at 50 °C with a nine-minute gradient using 0.2 mM ammonium fluoride in water and methanol.
• Mass spectrometry in negative-ion heated electrospray mode with selected reaction monitoring of two transitions per analyte and stable-isotope internal standards; cycle time 0.4 seconds.
• Data acquisition and processing via TraceFinder software version 4.1.

Used Instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific TSQ Altis triple-stage quadrupole mass spectrometer with HESI source
• Thermo Scientific Accucore Bi-Phenyl column, 2.6 µm, 50 × 2.1 mm

Results and Discussion

• Limit of quantitation of 2 pg/ml for both estrone and estradiol using 200 µl of matrix, with potential to reach 1 pg/ml at higher sample volume.
• Calibration curves linear across the tested range with ion ratio confirmation within ±20%.
• Precision: intra- and inter-day RSDs below 8.5% for estrone and below 7.9% for estradiol at levels of 5, 20, and 200 pg/ml.
• Recoveries between 105% and 122% across ten lots of human matrix and surrogate BSA matrix.
• Minimal matrix effects observed for internal standards in ten CDC HoSt samples.
• Accuracy: measured values within ±15% of CDC reference concentrations except one sample at 24% deviation.

Practical Benefits and Applications

• No derivatization reduces analysis time and cost
• High sensitivity and specificity suitable for low-level steroid profiling
• Robust performance supports large-scale clinical and research studies

Future Trends and Potential Applications

• Extension to additional steroid hormones and metabolites
• Further lowering detection limits through larger sample volumes and enhanced ionization techniques
• Automation of sample preparation and data processing for higher throughput
• Application to population studies and personalized hormone therapy monitoring

Conclusion

The developed LC-MS/MS assay provides sensitive, accurate, and reproducible measurement of estrone and estradiol in human serum without derivatization, enabling improved clinical research into low-abundance steroid hormones.

References

1. Rosner W; Hankinson SE; Sluss PM; Vesper HW; Wierman ME Challenges to the measurement of estradiol an Endocrine Society position statement J Clin Endocrinol Metab 2013 8 1376–1387
2. Rosner W; Auchus RJ; Azziz R; Sluss PM; Raff H Utility limitations and pitfalls in measuring testosterone an Endocrine Society position statement J Clin Endocrinol Metab 2007 92 405–413
3. Soldin SJ; Soldin OP Steroid hormone analysis by tandem mass spectrometry Clin Chem 2009 55 1061–1066