USP Method Transfer of Levonorgestrel and Ethinyl Estradiol Tablets from HPLC to UPLC

Importance of the Topic

Chromatographic assays for generic drug quality rely on standardized USP monograph methods. Traditional HPLC methods using 5 µm columns often suffer from long run times, high solvent consumption and frequent column replacement. Transitioning these assays to UPLC with sub-2 µm particles offers substantial time savings, reduced solvent and sample usage, and enhanced laboratory productivity, which is critical for routine analysis of formulations such as levonorgestrel and ethinyl estradiol tablets.

Objectives and Overview of the Study

The study aimed to assess and transfer the USP compendial method for levonorgestrel and ethinyl estradiol tablets from HPLC to UPLC. Key goals were:

• Evaluate two L7-designated HPLC columns (Zorbax C8 and XBridge C8) under the USP isocratic method.
• Scale the method to UPLC using an ACQUITY UPLC BEH C8 column via the UPLC Columns Calculator.
• Compare system suitability metrics, throughput, solvent and sample consumption.
• Examine column performance and pressure trends over extended injections.
• Develop a cleaning strategy to mitigate pressure increases during routine use.

Methodology and Instrumentation

• HPLC System: Waters Alliance 2695 with Zorbax C8 and XBridge C8 columns (4.6×150 mm, 5 µm), isocratic mobile phase 7:3:9 acetonitrile:methanol:water, 1 mL/min, 50 µL injection, UV detection at 215 nm.
• UPLC System: Waters ACQUITY UPLC H-Class equipped with BEH C8 column (2.1×50 mm, 1.7 µm), scaled flow 0.61 mL/min, 3.5 µL injection, same mobile phase and detection.
• Data Management: Empower 2 CDS and Waters UPLC Columns Calculator for method scaling.
• Sample Preparation: Tablet dissolution targeting 15 µg/mL levonorgestrel and 3 µg/mL ethinyl estradiol, sonication, mechanical shaking, centrifugation at 4000 rpm then 12000 rpm to yield clear supernatant.

Main Results and Discussion

Both HPLC columns met USP resolution (NLT 2.5) and peak area RSD (NMT 2.0 %) criteria, with the XBridge C8 column delivering faster elution. The UPLC transfer reduced run time from 10 min to 1.5 min (85 % decrease) and cut solvent/sample usage by 92 %, while maintaining resolution above 5.3 and RSD well below 2 %. A routine use study on UPLC demonstrated sustained performance over 2200 injections, with resolution remaining above 3.3 and peak area RSD below 1 %. Backpressure increased by 36 % on UPLC versus 74 % on HPLC over 1000 injections. Incorporating a post-run gradient wash to 100 % acetonitrile stabilized pressure throughout extended use.

Benefits and Practical Applications

• High throughput analysis enables rapid QC testing of generic formulations.
• Significant solvent and sample savings reduce operational costs and environmental impact.
• Enhanced column lifetime and robustness through gradient washing.
• Alignment with USP monograph ensures regulatory compliance and method standardization.

Future Trends and Opportunities

Wider adoption of UPLC for method updates across diverse drug classes is anticipated, along with automated column cleaning protocols to further minimize pressure build-up. Integration of advanced data analytics and AI-guided method optimization could streamline method transfer workflows and enhance routine QC efficiency.

Conclusion

The USP monograph assay for levonorgestrel and ethinyl estradiol was successfully transferred from HPLC to UPLC using matched column chemistries and scaling tools. The UPLC method delivered dramatic improvements in speed, resource consumption and long-term robustness. A strategic gradient wash step ensures stable column performance during extended routine use, making this approach highly suitable for high-throughput quality control environments.

Reference

• USP Monograph. Levonorgestrel and Ethinyl Estradiol Tablets, USP34-NF29, United States Pharmacopeial Convention, 2011.
• Jones M.D., Alden P., Fountain K.J., Aubin A. Implementation of Methods Translation between Liquid Chromatography Instrumentation, Waters Application Note, 2010, Part No. 720003721EN.