Highly Sensitive and Robust Quantification Method for Ethinyl Estradiol and Drospirenone in Plasma

Importance of the Topic

Combined ethinyl estradiol and drospirenone formulations are among the most widely used low-dose oral contraceptives due to their efficacy and safety. Ethinyl estradiol exhibits low bioavailability because of extensive metabolism and high protein binding, while drospirenone is fully metabolized to inactive products and eliminated via hepatic and renal pathways. Accurate quantification at sub-picogram levels is critical in bioequivalence and pharmacokinetic evaluations of these compounds.

Objectives and Study Overview

The aim of this work was to establish and validate a highly sensitive, robust and reproducible liquid chromatographic tandem mass spectrometric method for simultaneous quantification of ethinyl estradiol and drospirenone in human plasma. The target lower limit of quantification for ethinyl estradiol was set at 1.0 pg/mL.

Methodology

500 μL plasma samples spiked with analyte and internal standards underwent liquid–liquid extraction using tert-butyl methyl ether/hexane, followed by evaporation and derivatization of ethinyl estradiol with dansyl chloride under basic conditions. A second extraction removed excess reagent. The final extract was reconstituted in acetonitrile/water for analysis. Chromatographic separation was achieved on a C18 50 × 2.1 mm column at 30 °C using a gradient of 5 mM ammonium formate buffer and acetonitrile:methanol at 0.3 mL/min with an 8-minute run time.

Instrumentation

• SCIEX Triple Quad 5500 LC-MS/MS system with Turbo V electrospray ion source in positive mode
• Shimadzu Nexera UPLC with C18 analytical column (50 × 2.1 mm, 5 μm)
• Valco switching valve for diversion of initial eluent

Main Results and Discussion

Dansyl derivatization enhanced ionization of ethinyl estradiol, generating a prominent m/z 530.2 → 171.1 transition. Drospirenone was monitored at m/z 367.1 → 97.0. Calibration curves were linear from 0.993 to 300.48 pg/mL for ethinyl estradiol (r = 0.9980) and from 1003.63 to 171152.24 pg/mL for drospirenone (r = 0.9994). The method achieved an LLOQ of 0.993 pg/mL for ethinyl estradiol (S/N 68) and 1003.63 pg/mL for drospirenone (S/N 284). Intra- and inter-batch precision and accuracy for both analytes met regulatory criteria with values between 80 and 120 percent across multiple quality control levels.

Benefits and Practical Applications

• High sensitivity at sub-picogram level supports bioequivalence and pharmacokinetic studies
• Simple liquid–liquid extraction and derivatization streamline sample preparation
• Short run time enhances throughput for regulated bioanalytical laboratories

Future Trends and Applications

Further improvements may involve ultrafast chromatography, application to additional biological matrices, multiplexing with other steroidal compounds and integration with high-resolution mass spectrometry. Automation of derivatization and extraction steps could increase sample throughput for large clinical studies.

Conclusion

A validated LC-MS/MS assay on the SCIEX Triple Quad 5500 provides a sensitive, accurate and reproducible approach for quantifying ethinyl estradiol and drospirenone in human plasma. It meets stringent requirements for bioanalytical laboratories and supports reliable pharmacokinetic and bioequivalence evaluations.

References

• Back DJ, Miners JO The gut wall metabolism of ethinyloestradiol and its contribution to the presystemic metabolism of ethinyloestradiol in humans Br J Clin Pharmacol 1982 13 325 330
• Rogers SM, Back DJ, Miners JO Paracetamol interaction with oral contraceptive steroids increased plasma concentrations of ethinyloestradiol Br J Clin Pharmacol 1987 23 721 725
• Guengerich FP, et al Metabolism of 17α-ethynylestradiol in humans Life Sci 1990 47 1981 1988
• Williams MC, et al The urinary metabolites of 17α-ethynylestradiol-9α,11β-³H in women Chromatographic profiling and identification of ethinyl and non-ethinyl compounds Steroids 1975 25 229 246
• Huber J, Spona J, Corbus M Efficacy and tolerability of a monophasic oral contraceptive containing ethinyl estradiol and drospirenone Eur J Contracept Reprod Health Care 2000 5 25 34
• Foster DG, et al Number of oral contraceptive pill packages dispensed method continuation and costs Obstet Gynecol 2006 108 1107 1114
• Anari MR, et al Derivatisation of ethinyl estradiol with dansyl chloride to enhance electrospray ionization application in trace analysis in rhesus monkey plasma Anal Chem 2002 74 16 4136 4144
• Eduardo AJ, et al Bioequivalence of two oral contraceptive drugs containing ethinyl estradiol and drospirenone in healthy female volunteers Global J Med Res 2011 11 3 9 17