Analysis of free plasma ethinyl estradiol

Importance of the topic

Accurate measurement of ethinyl estradiol at picogram levels in human plasma underpins clinical pharmacology, contraceptive efficacy studies and endocrine research. The ability to quantify free ethinyl estradiol with high sensitivity and minimal matrix interference is essential for reliable bioanalysis and regulatory compliance.

Study objectives and overview

This work aimed to develop and validate a sample preparation and LC-MS/MS protocol capable of detecting ethinyl estradiol down to 5 pg/mL in human plasma. Key goals included optimizing the new SOLA 30 mg SCX solid phase extraction phase, minimizing matrix effects, maximizing recovery and demonstrating rapid chromatographic separation.

Methodology and analytical workflow

The analytical procedure comprised the following steps:

1. Plasma sample loading: 1 mL of human plasma was spiked with analyte and internal standard and diluted with ammonium formate buffer.
2. SPE clean-up: Wells of the SOLA SCX 30 mg plate were conditioned, samples loaded, washed with water/methanol mixtures and eluted with methanol.
3. Derivatization: Dried extracts were reconstituted in sodium bicarbonate buffer, reacted with dansyl chloride at 60 °C for 30 minutes to enhance ionization.
4. UHPLC-MS/MS analysis: Dansylated analytes were separated by isocratic elution (80% acetonitrile) on a Hypersil GOLD Vanquish C18 column with a 1.5-minute run time, and quantified using TSQ Altis triple quadrupole mass spectrometry in positive H-ESI mode.

Instrumental setup

• Vanquish Horizon UHPLC system with binary pump, split sampler, active preheater and column compartment.
• Hypersil GOLD Vanquish C18 column (100 × 2.1 mm, 1.9 µm).
• TSQ Altis triple quadrupole mass spectrometer with positive heated electrospray ionization.
• Thermo Scientific Chromeleon CDS software for control and data processing.

Results and discussion

The method achieved a lower limit of quantitation of 5 pg/mL with linearity up to 200 pg/mL (r²=0.993). Calibration standards showed bias within ±11%, and quality control samples exhibited accuracy and precision below 15%. Matrix effects were minimal (normalized factor 1.03) due to the selectivity of the SCX sorbent. Mean recovery at the mid QC level was 91.3% for ethinyl estradiol and 88.4% for the deuterated internal standard. Peak shapes remained sharp at low concentrations, enabling a rapid 1.5-minute analysis.

Benefits and practical application

• High throughput: 96-well SPE format and fast isocratic UHPLC shorten turnaround time.
• Robustness: Fritless polymeric sorbent reduces blockages, improving reproducibility for viscous plasma samples.
• Enhanced sensitivity: Dansyl derivatization boosts ionization efficiency, lowering LOQ to 5 pg/mL.
• Versatility: Applicable to bioanalytical and clinical studies requiring trace estrogen quantitation.

Future trends and potential applications

Emerging directions include automated SPE workflows, multiplexed analysis of steroid hormones, integration with high-resolution mass spectrometry and application to large-scale clinical trials. Advances in sorbent chemistries and derivatization strategies may further improve sensitivity and selectivity for challenging analytes.

Conclusion

This application demonstrates a highly sensitive, reproducible and fast LC-MS/MS method for free ethinyl estradiol quantitation in human plasma. The combination of SOLA SCX SPE, dansyl derivatization and rapid isocratic UHPLC delivers excellent recovery, low matrix effects and throughput suitable for demanding bioanalytical applications.

Reference

• Wright K P, Johnson J V. Evaluation of extended and continuous use oral contraceptives. Therapeutics and Clinical Risk Management. 2008;4(5):905–911.