USP Analysis of Norethindrone and Mestranol Tablets with Agilent Poroshell 120 EC-CN and EC-C8 Columns

Importance of the Topic

High-performance liquid chromatography (HPLC) methods for steroidal tablets, such as those containing norethindrone and mestranol, require robust, efficient separations to meet pharmacopeial standards. Transitioning from traditional fully porous 5 µm particles to superficially porous (Poroshell) columns can dramatically shorten analysis times, reduce solvent consumption, and maintain or improve chromatographic performance while complying with United States Pharmacopeia (USP) Chapter 621.

Objectives and Study Overview

This study aimed to transfer two USP methods (dissolution and assay) for norethindrone and mestranol tablets from recommended 5 µm columns to Agilent Poroshell 120 superficially porous columns (2.7 µm), then evaluate system suitability, resolution, precision, and efficiency under modified conditions that remain within USP guidelines.

Methodology and Instrumentation

• Columns used
  
  • Agilent ZORBAX Eclipse XDB-CN, 4.6×250 mm, 5 µm (USP reference)
  • Agilent Poroshell 120 EC-CN, 3.0×100 mm, 2.7 µm
  • Agilent ZORBAX Eclipse XDB-C8, 4.6×150 mm, 5 µm (USP reference)
  • Agilent Poroshell 120 EC-C8, 4.6×50 mm, 2.7 µm
• Instrumentation
  
  • Agilent 1290 Infinity LC system enhanced with Ultra-Low Dispersion kit (0.075 mm id capillaries)
  • Detector wavelengths: 205 nm for CN methods, 200 nm for C8 methods
• Chromatographic conditions
  
  • Isocratic elution (40 % acetonitrile for dissolution; 50 % acetonitrile for assay)
  • Flow rates adjusted: up to 2 mL/min on shorter columns
  • Column temperature maintained at 25 °C

Main Results and Discussion

• Dissolution analysis on CN phases
  
  • USP 5 µm column: 18 min run time; baseline separation of norethindrone, progesterone (internal standard), mestranol
  • Poroshell 120 EC-CN at optimized flow: separation in 2.5 min
  • System suitability: RSDs < 1 % on shorter columns; theoretical plates > 14 000; tailing factors < 1.0
• Assay analysis on C8 phases
  
  • USP 5 µm column: 16 min run time; resolution P-M > 11
  • Poroshell 120 EC-C8 at 2 mL/min: complete separation in < 2.5 min
  • System suitability: RSDs < 1.5 %; plates > 10 000; resolution > 10.8

Benefits and Practical Applications

• Run time reduction up to 85 %, boosting sample throughput
• Solvent consumption decreased by similar factor, lowering operational costs
• Maintained or improved chromatographic performance within USP guidelines
• Compatibility with standard LC systems due to moderate backpressure of superficially porous particles

Future Trends and Applications

• Broader adoption of superficially porous columns for pharmacopeial methods across diverse APIs
• Integration with UHPLC and high-throughput screening to further shorten analysis times
• Development of green chromatography workflows via reduced solvent use
• Application in complex formulations and multi-component assays

Conclusion

Agilent Poroshell 120 superficially porous columns effectively replace traditional 5 µm columns for USP dissolution and assay of norethindrone and mestranol tablets. The shorter columns deliver significant time and cost savings while fully complying with USP system suitability requirements, offering a practical route to higher laboratory efficiency.

References

1. A Gratzfeld-Hüsgen and E Naegele, Maximizing efficiency using Agilent Poroshell 120 columns, Application Note, Agilent Technologies Inc., publication 5990-5602EN, 2010
2. V R Meyer, Practical High Performance Liquid Chromatography, 4th ed., p 34, Wiley, 2004
3. A Mack, Optimizing Performance of an Agilent ZORBAX RRHD Eclipse Plus C18 Column by Enhancing an Agilent 1290 Infinity LC System for Ultra-Low Dispersion, Application Note, Agilent Technologies Inc., publication 5990-9502EN, 2011