Analysis of Estrone and Estradiol to Low pg/mL Levels in Human Serum by Triple Quadrupole Mass Spectrometry for Clinical Research

Significance of the Topic

Quantifying estrone and estradiol at ultra-low picogram-per-milliliter concentrations in human serum is essential for clinical research in endocrinology, hormone therapy monitoring and epidemiological studies. Sensitive and specific measurement supports accurate assessment of hormonal status and standardization across laboratories.

Objectives and Study Overview

This work describes the development and validation of an LC-MS/MS method using a triple quadrupole mass spectrometer to measure estrone and estradiol in human serum. The aim was to achieve reliable detection down to 2 pg/mL, evaluate precision and accuracy over multiple days, and confirm method robustness with CDC Hormone Standardization (HoSt) Program samples.

Methodology

Samples (200 µL) underwent liquid-liquid extraction with tert-butyl methyl ether, evaporation and reconstitution in 30 % methanol. Reversed-phase chromatographic separation was performed with a nine-minute gradient. Detection employed selected reaction monitoring transitions for analytes and isotopically labeled internal standards, with heated electrospray ionization.

Used Instrumentation

• Thermo Scientific Vanquish Horizon HPLC with Accucore Bi-Phenyl column (2.6 µm, 50 × 2.1 mm) heated to 50 °C
• Thermo Scientific TSQ Altis triple quadrupole mass spectrometer equipped with HESI source
• Thermo Scientific Trace Finder software version 4.1 for data acquisition and processing

Main Results and Discussion

The method showed a limit of quantitation of 2 pg/mL for both estrone and estradiol using 200 µL serum, with potential to reach 1 pg/mL at 500 µL. Inter-day precision across QC levels (5, 20, 200 pg/mL) yielded RSDs below 8.5 %. Accuracy testing with CDC HoSt samples demonstrated 90 % of values within ±15 % of reference concentrations (one sample within 24 %). Recovery of internal standards across ten different serum and plasma lots ranged from 107 % to 119 %, indicating consistent extraction. Matrix effects were negligible, confirming minimal interference from endogenous components.

Benefits and Practical Applications

• Enables accurate quantitation of ultra-low steroid levels for clinical and research laboratories
• Aligns with CDC HoSt program requirements for hormone standardization
• Provides reproducible and precise data for patient monitoring, bioequivalence and epidemiological studies

Future Trends and Applications

Future developments may focus on automating sample preparation to increase throughput, employing microflow or nanoflow LC to enhance sensitivity, and extending the method to additional steroid hormones. Advances in mass spectrometer design could further lower detection limits and support personalized medicine research.

Conclusion

This study demonstrates a robust, sensitive LC-MS/MS assay for estrone and estradiol in human serum at picogram levels. The method’s precision, accuracy and minimal matrix effects make it well suited for demanding clinical research applications.

References

CDC Hormone Standardization (HoSt) Program Phase 1 samples
Thermo Fisher Scientific product and application documentation