Determination of Gestodene in Human Plasma by SLE-LC-MS/MS Using a Solid Core HPLC Column

Significance of the Topic

Gestodene quantification in human plasma is essential for pharmacokinetic and bioequivalence studies of oral contraceptives containing this synthetic progestin. Reliable measurement supports dosing optimization, therapeutic monitoring, and regulatory compliance. Advances in sample preparation and chromatographic separation can streamline workflows in clinical and research laboratories.

Aims and Study Overview

The study aimed to develop and validate a rapid, sensitive LC-MS/MS method combining solid-supported liquid-liquid extraction (SLE) on HyperSep SLE cartridges with separation on an Accucore C18 core-shell column. Deuterated gestodene (d6-gestodene) served as an internal standard to ensure accuracy and precision across a dynamic range of 0.05–5 ng/mL.

Methodology and Instrumentation

Sample preparation employed SLE with a diatomaceous earth support to isolate gestodene from 300 μL of plasma. Following acidification, analytes were eluted with methyl tert-butyl ether, evaporated, and reconstituted in methanol/water (80:20). Chromatographic separation was achieved on a Thermo Scientific Accucore C18 column (50×2.1 mm, 2.6 μm) at 40 °C, using a gradient of 5–95% methanol with 0.1% formic acid over 5 minutes at 0.4 mL/min. MS detection utilized a TSQ Vantage triple quadrupole with heated electrospray ionization in positive mode, employing SRM transitions m/z 311.2→109.1 for gestodene and m/z 317.2→114.1 for d6-gestodene.

Used Instrumentation

• Thermo Scientific HyperSep SLE 500 mg/3 mL cartridges
• Accucore C18 core-shell column (50×2.1 mm, 2.6 μm)
• Thermo Scientific Accela 1250 HPLC system with Open Autosampler
• Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer with HESI-II source

Main Results and Discussion

• Retention time of gestodene: 2.84 minutes, with sharp, symmetrical peaks and minimal tailing.
• Linearity: 0.05–5 ng/mL, r² = 0.999, with relative errors within ±5% across calibrants.
• Precision: mid-range QC (2.5 ng/mL) showed <2% CV (n=6), meeting FDA bioanalytical validation criteria.
• Sensitivity: lower limit of quantification at 0.05 ng/mL, enabling detection at low endogenous levels.
• Specificity: SRM chromatograms demonstrated clean background and clear analyte peaks without interference.

Benefits and Practical Applications

• Reduced solvent usage and elimination of emulsions compared to traditional LLE.
• Automatable workflow compatible with standard SPE manifolds, enhancing throughput and consistency.
• Small plasma volume requirement (300 μL) suits limited sample availability.
• Core-shell column technology ensures fast separations and high efficiency, reducing analysis time to 5 minutes per sample.
• Robust validation supports routine bioanalytical testing in pharmacokinetic, clinical, and QA/QC laboratories.

Future Trends and Potential Uses

• Integration of microflow LC to further reduce solvent consumption and enhance sensitivity.
• Expansion to multiplexed hormone panels using similar SLE-LC-MS/MS protocols.
• Automation of SLE on robotic platforms for high-throughput clinical studies.
• Implementation of advanced core-shell and superficially porous phases for faster, greener chromatography.

Conclusion

The developed SLE-LC-MS/MS method offers a robust, rapid, and sensitive approach for quantifying gestodene in human plasma. Core-shell column technology paired with SLE extraction ensures high throughput, minimal solvent use, and compliance with regulatory validation standards, making it well-suited for routine bioanalytical applications.

References

1. Junior EA, Duarte LF, Pirasol Vanunci ML, Teixeira ML. Bioequivalence of Two Oral Contraceptive Drugs Containing Ethinylestradiol and Gestodene in Healthy Female Volunteers. J Bioequiv Availab. 2010;2:125–130. doi:10.4172/jbb.1000044
2. U.S. Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. October 1, 2012.