A Simple Conversion of the USP Assay Method for Diphenhydramine HCl to the Agilent InfinityLab Poroshell 120 Column EC-C8

Significance of the Topic

Pharmaceutical laboratories rely on compendial methods to ensure consistent testing of raw materials and drug products. Adapting existing USP assay methods to modern superficially porous columns can substantially reduce analysis time and solvent consumption while maintaining compliance with system suitability criteria. Such improvements support higher throughput, lower operational costs, and greener analytical workflows without the need for major instrument upgrades.

Objectives and Study Overview

This application note demonstrates the conversion of the USP monograph assay for diphenhydramine hydrochloride from a 4.6×250 mm, 5 µm C8 column to an Agilent InfinityLab Poroshell 120 EC-C8 column (4.6×100 mm, 2.7 µm). The goal is to shorten the assay time from 13 to approximately 3.5 minutes by adjusting flow rates and gradient times under the forthcoming USP Stage 4 Harmonization guidelines, while satisfying all system suitability requirements.

Methodology

Samples of diphenhydramine HCl and its related impurity were prepared in a mixture of potassium phosphate buffer (pH 3.0) and acetonitrile (35:65). The original USP gradient method was scaled according to column dimensions and flow rates, using the formula tG2 = tG1 × (L2 × dC2²)/(L1 × dC1²) × (F1/F2). Flow rates from 1.2 to 1.8 mL/min were evaluated to identify the optimum condition that retained the (t/Wh)2 ratio within −25% to +50% of the USP reference. The detection wavelength was set at 220 nm, column temperature at 25 °C, and injection volume scaled accordingly.

Used Instrumentation

• Agilent 1260 Infinity II LC System
• Binary Pump (G7117B), Multisampler (G7167A), Multicolumn Thermostat (G7116A), Diode Array Detector (G7117A)
• Agilent ZORBAX Eclipse Plus C8, 4.6×250 mm, 5 µm
• Agilent InfinityLab Poroshell 120 EC-C8, 4.6×100 mm, 2.7 µm
• Standard LC consumables: 0.17 mm tubing, 2 mL amber vials, heater and 1 µL flow cell

Main Results and Discussion

The transfer achieved a reduction in run time from 13 to 3.5 minutes at 1.8 mL/min without exceeding instrument pressure limits. System suitability parameters for resolution (2.96, NLT 1.5), tailing factor (1.11, NMT 1.8), and %RSD for six injections (0.28% for resolution solution, 0.68% for area) were met. The (t/Wh)2 metric remained between 1,808 and 3,615 across tested flows, confirming equivalence in chromatographic performance.

Benefits and Practical Applications

• Analysis time cut by over 70%, increasing daily sample throughput.
• Savings in solvent consumption and reduced environmental impact.
• No need for new instrumentation or full revalidation under USP Stage 4 guidelines.
• Maintained compendial compliance and consistent impurity resolution.

Future Trends and Potential Applications

As USP harmonization advances, more monograph methods can be converted to superficially porous and sub-2 µm columns, further enhancing efficiency. Integration with ultra-high-pressure systems, automated method transfer tools, and green chemistry initiatives will drive continued optimization. Artificial intelligence could support predictive scaling and real-time method adjustment across multi-site operations.

Conclusion

This study confirms that the USP assay for diphenhydramine HCl can be successfully transferred to an Agilent InfinityLab Poroshell 120 EC-C8 column, achieving significant reductions in run time and solvent use while meeting all compendial system suitability criteria. The approach requires no major hardware changes, offering an efficient path to modernize quality-control assays.

References

1. United States Pharmacopeia 43(4). USP Diphenhydramine HCl, Impurity Method.
2. United States Pharmacopeia, General Chapter <621>, USP 37-NF32, First Supplement.
3. USP Harmonized Standards, Supplement USP Stage 4 Harmonization, Official December 1, 2022.