USP Analysis of Diphenhydramine and Pseudoephedrine Using an Agilent Poroshell 120 EC-CN Column

Significance of the Topic

Today's pharmaceutical quality control relies heavily on reliable and efficient chromatographic methods to quantify active ingredients in dosage forms. The United States Pharmacopeia (USP) method for analyzing diphenhydramine and pseudoephedrine capsules is widely adopted, yet it traditionally employs a 5 µm fully porous column that entails longer run times and higher solvent usage. Transitioning to superficially porous particle technology offers the potential to reduce analysis time, lower solvent consumption, and maintain compliance with USP guidelines.

Objectives and Study Overview

This study aimed to adapt the USP assay for diphenhydramine and pseudoephedrine capsules by replacing the specified 4.6 × 250 mm, 5 µm column with a shorter 3.0 × 100 mm, 2.7 µm superficially porous Agilent Poroshell 120 EC-CN column. The goals were to verify that the modified method met all USP Chapter 621 requirements and to quantify time and solvent savings achieved by the new column format.

Methodology and Instrumentation

The method employed an Agilent 1290 Infinity LC system equipped with an Ultra Low Dispersion Kit to minimize extra-column volume. Chromatographic conditions were:

• Mobile phase A: 10 mM heptanesulfonate, 13 mM triethylamine, pH 3.3
• Mobile phase B: acetonitrile:methanol (26:10, v/v); isocratic 36 % B
• Column temperature: 25 °C
• Detection: UV at 254 nm
• Flow rates: 2 mL/min on the 5 µm column; 0.85 mL/min on the Poroshell 120 column
• Injection volumes: 20 µL and 3.5 µL, respectively

Main Results and Discussion

Both columns achieved baseline separation of pseudoephedrine and diphenhydramine with resolution values well above the USP minimum of 3.0 (Rs 24.3 for the 5 µm column; Rs 21.2 for the Poroshell 120). Tailing factors remained below 1.6, and reproducibility for replicate injections was within 2.0 % RSD for both analytes. Retention time shifts were consistent with column length and particle size differences, demonstrating predictable performance upon method transfer. The Poroshell 120 column reduced the total analysis time from approximately 5.5 minutes to 1.5 minutes, cutting solvent use by more than half.

Benefits and Practical Applications of the Method

The introduction of superficially porous particles significantly enhances laboratory throughput by shortening run times and reducing solvent consumption. Laboratories performing routine QC on diphenhydramine/pseudoephedrine formulations can adopt this approach without regulatory hurdles, as all modifications fall within USP Chapter 621 allowances. Cost savings from decreased solvent use and increased sample throughput further improve operational efficiency.

Future Trends and Potential Applications

Advances in superficially porous particle manufacturing continue to push the boundaries of high-throughput LC analysis. Future developments may include broader adoption of shorter columns with similar benefits for other pharmacopeial assays, integration with ultra-high-pressure systems for even faster separations, and expansion into mass spectrometry–compatible mobile phases to enhance sensitivity and selectivity.

Conclusion

Substituting the USP-specified 5 µm column with a 2.7 µm superficially porous Agilent Poroshell 120 EC-CN column successfully fulfills all chromatographic criteria for the assay of diphenhydramine and pseudoephedrine capsules. This modification delivers substantial gains in speed, solvent economy, and lab productivity while maintaining compliance with regulatory standards.

References

1. A. Gratzfeld-Hüsgen, E. Naegele. Maximizing efficiency using Agilent Poroshell 120 columns. Agilent Technologies Application Note 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. Agilent Technologies Application Note 5990-9502EN (2011).