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

Importance of the Topic

Routine pharmaceutical analysis relies heavily on compendial HPLC methods defined by the U.S. Pharmacopeia. Converting these well-established assays to modern superficially porous particle columns, such as Agilent InfinityLab Poroshell 120 EC-C8, enables significant reductions in run time and solvent consumption while maintaining resolution on existing LC instrumentation.

Goals and Study Overview

This application note demonstrates the transfer of the USP assay for benzocaine lozenges—originally performed on a 5 µm, 4.6×250 mm ZORBAX Eclipse Plus C8 column—to shorter InfinityLab Poroshell 120 EC-C8 columns (150 mm, 100 mm, and 75 mm) with 4 µm and 2.7 µm particles, following USP <621> permitted adjustment rules without revalidation.

Methodology and Instrumentation

• Instrument configuration: Agilent 1260 Infinity II LC system with binary pump, multisampler, multicolumn thermostat, and diode array detector (280 nm).
• Columns evaluated: ZORBAX Eclipse Plus C8 (4.6×250 mm, 5 µm) and InfinityLab Poroshell 120 EC-C8 (4.6×150, 4.6×100, 4.6×75 mm; 4 µm and 2.7 µm superficially porous particles).
• Mobile phase: premixed acetonitrile : water : 1.0 M KH₂PO₄ buffer (pH 3.0) in a 200 : 750 : 50 mL ratio.
• Flow rate maintained at 1.5 mL/min, column temperature at 25 °C, and injection volumes scaled geometrically between 6 µL and 20 µL.
• Sample and standard preparation followed USP guidelines using Diluent A (0.1 N HCl) and Diluent B (50:50 acetonitrile:water) with benzocaine reference standard at 0.01 mg/mL.

Main Results and Discussion

• 150 mm columns (4 µm and 2.7 µm) provided ~40% faster runs, reducing retention from ~15 min to ~6.4 min, with operating pressures of 264–421 bar.
• 100 mm and 75 mm, 2.7 µm Poroshell 120 EC-C8 columns achieved 60% and 70% time savings, yielding retention times of ~6.4 min and ~4.5 min at 314 bar and 287 bar, respectively.
• All shortened methods met USP system suitability criteria: theoretical plates within ±25–50% of the original, tailing factor ≤1.5, and RSD ≤2.0% without further validation.
• Solvent usage decreased in direct proportion to analysis time, offering up to 70% reduction in mobile phase consumption.

Benefits and Practical Applications

Adopting superficially porous particle columns allows QC laboratories to increase sample throughput, reduce solvent costs, and maintain existing LC hardware, providing flexible scheduling and improved productivity for routine compendial testing.

Future Trends and Potential Applications

Growth in superficially porous and sub-2 µm stationary phases will drive wider adoption of UHPLC technology. Emerging trends include ultra-high-pressure operation, green solvent strategies, method miniaturization, and integration with automated data workflows and laboratory information management systems for enhanced efficiency and sustainability.

Conclusion

The USP benzocaine lozenge assay can be seamlessly transferred to shorter Agilent InfinityLab Poroshell 120 EC-C8 columns under USP <621> guidelines, achieving up to 70% faster analysis and solvent savings without compromising chromatographic performance or requiring revalidation.

Reference

1. United States Pharmacopeia. USP Benzocaine Lozenges Method. USP 42–NF 37, 2020.
2. United States Pharmacopeia. General Chapter <621> Chromatography. USP 37–NF 32, Supplement 1.