High-Throughput LC-MS/MS Quantification of Estrone (E1) and Estradiol (E2) in Human Blood Plasma/Serum for Clinical Research Purposes

Importance of the Topic

Estrone and estradiol are essential steroid hormones governing female reproductive physiology and are critical biomarkers in clinical and research settings. Accurate and sensitive quantification in plasma or serum supports studies of menstrual cycle regulation, endocrine disorders, and therapeutic interventions.

Objectives and Study Overview

This work aimed to develop and validate a high-throughput LC-MS/MS method for reliable measurement of dansylated estrone and estradiol in human blood plasma and serum over a range of 5 to 500 pg/mL. The study included method optimization, performance evaluation, and comparison with an independent reference laboratory.

Methodology and Instrumentation

• Sample Preparation: Liquid–liquid extraction with methyl t-butyl ether followed by dansyl chloride derivatization to enhance positive-ion formation.
• Chromatography: Transcend LX-4 HPLC with Accucore RP-MS column (50 × 2.1 mm, 2.6 µm) at 40 °C using a water/methanol gradient with 0.1% formic acid.
• Mass Spectrometry: TSQ Endura triple quadrupole with heated electrospray ionization; SRM transitions monitored for quantitation and confirmation of analytes and deuterated internal standard.
• Data Analysis: Thermo Scientific TraceFinder software for peak integration, calibration, and ion-ratio confirmation.

Main Results and Discussion

• Calibration linearity from 5 to 500 pg/mL achieved with r2 ≥ 0.995 (1/X weighting).
• Intra- and inter-batch precision better than 6% and 7% CV, respectively; carryover below 0.5%.
• Ion ratio confirmation within 20% of calibrator averages, supporting high specificity.
• Internal standard recovery averaged 37% across samples, fully compensating matrix effects.
• Comparison with reference laboratory data for 40 donor samples demonstrated 95% of results within ±20% difference.

Benefits and Practical Applications

• High sensitivity allows quantitation at low picogram per milliliter levels for clinical research.
• Robust precision and minimal carryover enable reliable high-throughput analysis.
• Multichannel HPLC configuration supports up to 40 injections per hour.
• Derivatization strategy improves ionization efficiency and selectivity for steroid profiling.

Future Trends and Applications

Emerging ultrahigh-performance chromatography and microflow LC-MS/MS approaches are expected to reduce run times and solvent consumption. New derivatization reagents and enhanced ion sources may offer improved sensitivity and lower sample volume requirements. Integration with automated sample preparation and laboratory information management systems will further streamline workflows. Applications in personalized medicine, pediatric endocrinology, and dynamic hormone monitoring are anticipated to expand.

Conclusion

The validated LC-MS/MS assay delivers sensitive, precise, and high-throughput quantitation of estrone and estradiol in plasma and serum. Its strong agreement with reference methods endorses its adoption in clinical research and quality control laboratories.

Used Instrumentation

• Thermo Scientific Transcend LX-4 HPLC system
• Accucore RP-MS column 50 × 2.1 mm, 2.6 µm
• Thermo Scientific TSQ Endura triple quadrupole MS with HESI probe
• Thermo Scientific TraceFinder data processing software

References

• Nelson R E et al Clin Chem 2004 50 373–384
• Kushnir M M et al Am J Clin Pathol 2008 129 530–539