Modernization of the Acetaminophen USP Monograph Gradient HPLC Method for Impurities using USP <621> Guidelines and MaxPeak Premier HPS Technology

Significance of the Topic

The modernization of United States Pharmacopeia (USP) gradient HPLC monograph methods addresses the need to update older column technologies, reduce analysis cycle time and solvent consumption, and accelerate quality control workflows in pharmaceutical laboratories.

Study Objectives and Overview

This work demonstrates the adaptation of the USP monograph method for organic impurities in acetaminophen to modern column technology under USP <621> guidelines. By replacing a 4.6×250 mm, 5 µm column and 73-minute gradient with a shorter 4.6×150 mm, 2.5 µm MaxPeak Premier HPS column and optimized gradient, the study aims to halve run time and reduce solvent use while meeting system suitability requirements.

Methodology and Used Instrumentation

• Original system: Agilent 1260 Infinity LC with UV detector; modernized system: ACQUITY Arc with 2998 PDA detector.
• Columns: Zorbax Eclipse Plus C8 (5 µm, 4.6×250 mm) versus XBridge Premier BEH C8 (2.5 µm, 4.6×150 mm).
• Detection at 254 nm; column temperature 40 °C; sample cooler 10 °C.
• Flow rates adjusted from 0.9 mL/min to 1.1 mL/min; injection volumes from 5 µL to 3 µL.
• Gradient profiles and method conditions calculated using Waters Column Calculator in line with USP <621> scaling formulas.

Main Results and Discussion

The modernized method produced comparable chromatographic performance: related compound tailing factors <1.2, resolutions >3.5, and peak area RSDs <1.0%. Run time decreased by 51% (from 73 to 36 minutes), and mobile phase usage fell by ~40% (from ~1.18 L to ~0.70 L). Batch analysis time for six replicate injections per standard and sample was reduced from ~22 hours to ~11 hours, doubling laboratory throughput.

Benefits and Practical Applications

• Accelerates release testing for generic and branded pharmaceuticals.
• Reduces operating costs through lower solvent consumption and instrument occupancy.
• Avoids need for ultra-high-pressure systems by using 2.5 µm particles compatible with conventional HPLC instruments.
• Offers a scalable column platform for future method adjustments across particle sizes.

Future Trends and Potential Applications

Continued evolution of column chemistries and digital calculation tools will simplify modernization of USP methods and support broader deployment of high-throughput LC workflows. Integration with automation and data-management systems will further enhance reliability and compliance.

Conclusion

By following USP <621> and leveraging the Waters Column Calculator, the acetaminophen impurity assay was modernized to deliver equivalent separation quality while halving analysis time and cutting solvent use by 40%, demonstrating a clear path for updating legacy monograph methods.

Reference

1. United States Pharmacopeia monograph for Acetaminophen.
2. USP General Chapter <621> Chromatography.