LC/MS/MS Method for Quantitative Determination of Ethinyl Estradiol in Human Plasma

Importance of the Topic

The quantification of ethinyl estradiol (EE) at ultra-low levels in human plasma is critical for pharmacokinetic studies, safety assessments of contraceptives and hormone therapies, and for monitoring therapeutic and toxicological thresholds. Achieving sub-picogram sensitivity enhances our ability to track EE exposure, supports dose optimization, and ensures patient safety in clinical and research settings.

Objectives and Overview of the Study

This work aimed to establish and validate an ultra-sensitive LC-MS/MS method for EE determination in plasma. Key goals included lowering the limit of quantitation to 1.0 pg/mL, ensuring linearity over a biologically relevant range (1–200 pg/mL), and demonstrating robust accuracy, precision, and selectivity against endogenous compounds.

Methodology

A two-stage sample preparation was developed:

• Liquid-liquid extraction: Plasma (750 µL) was acidified and extracted with hexane/MTBE (50:50), isolating parent and deuterated internal standard (EE-D4).
• Off-line derivatization: The extract was treated with dansyl chloride in alkaline buffer at 60 °C to introduce a tertiary amino group for enhanced ESI response.
• SPE cleanup: Dansylated extracts were loaded onto a mixed-mode SPE cartridge (Sola CX) to remove phospholipids and interferences before reconstitution and injection.

Used Instrumentation

The analytical platform consisted of a UHPLC Nexera system coupled to a Shimadzu LCMS-8040 triple quadrupole mass spectrometer with electrospray ionization. Chromatographic separation employed a Purospher STAR RP-18 column (100 mm × 2.1 mm, 2 µm) and a gradient of 0.1% formic acid in water and acetonitrile at 300 µL/min.

Main Results and Discussion

The method achieved a lower limit of quantitation of 1.0 pg/mL with a coefficient of variation of 15.1%. Calibration was linear from 1 to 200 pg/mL (R² = 0.9988) using weighted (1/x²) regression. Quality control samples at low, mid, and high levels (3, 90, 180 pg/mL) showed precision (%CV) ≤9.8% and accuracy within 91–104%. Average recovery of EE across QC levels was 64.5%, consistent and reproducible.

Benefits and Practical Applications

This validated assay supports:

• Pharmacokinetic and bioavailability studies of EE-containing products.
• Therapeutic drug monitoring and safety evaluation in clinical trials.
• Regulatory bioanalysis where ultra-low detection limits are required.

Future Trends and Potential Applications

Advances may include micro-sampling techniques (dried blood spots), automation of derivatization and SPE, and high-throughput platforms. Emerging high-resolution MS could further improve selectivity for complex matrices and facilitate multiplexed steroid profiling.

Conclusion

The presented LC-MS/MS method delivers robust, reproducible quantification of EE in human plasma at sub-picogram levels. Its sensitivity, combined with stringent cleanup, makes it a valuable tool for pharmacokinetic research and clinical bioanalysis.

References

• Drug Discov Ther 2007;1(2):108–118
• Rapid Commun Mass Spectrom 2004;18:1621–1628
• Biomed Chromatogr 2004;18:414–421
• J Chromatogr B 2005;825:223–232