Modernizing the USP Acetaminophen and Caffeine Tablets HPLC Method Following the Revised USP <621> Guidelines

Significance of the Topic

The modernization of pharmacopeial HPLC methods aligns with evolving column technologies to improve efficiency, reduce analysis time and solvent consumption, and maintain compliance with USP <621> guidelines. Adapting existing assays for acetaminophen and caffeine tablets illustrates practical benefits for quality control laboratories under resource and time constraints.

Objectives and Study Overview

This study aimed to update the USP monograph HPLC method for acetaminophen and caffeine tablets by:

• Replacing a conventional 4.6 × 100 mm, 5 µm totally porous particle (TPP) column with smaller particle alternatives.
• Applying 3.5 µm ZORBAX Eclipse Plus C18 and 4 µm InfinityLab Poroshell 120 EC-C18 columns.
• Ensuring system suitability requirements were met without full method revalidation.

Methodology

An isocratic separation was employed, with method adjustments guided by USP <621> equations:

• Flow rate adjustment (Equation 1) to compensate for changes in column inner diameter and particle size.
• Injection volume adjustment (Equation 2) to preserve chromatographic peak shapes.
• Maintenance of an acceptable column length-to-particle diameter (L/dp) ratio within −25 % to +50 % of the original method.

Used Instrumentation

The following instrumentation supported method evaluation:

• Agilent 1260 Infinity II LC system with binary pump, multisampler, column thermostat, and diode array detector.
• Columns evaluated: ZORBAX Eclipse Plus C18 (4.6 × 100 mm, 5 µm and 4.6 × 75 mm, 3.5 µm) and InfinityLab Poroshell 120 EC-C18 (4.6 × 50 mm, 4 µm).
• Mobile phase: methanol, glacial acetic acid, water (28:3:69); temperature: 45 °C; detection wavelengths: 275 nm (TPP) and 254 nm (SPP).

Main Results and Discussion

Both modernized columns met USP system suitability criteria:

• Tailing factors ≤ 1.2 for acetaminophen, caffeine, and benzoic acid.
• Resolution ≥ 1.4 between analyte and internal standard peaks.
• Peak area RSD ≤ 2 % (n=5).

Chromatograms confirmed sharp, well-resolved peaks.
Compared to the original method, the 3.5 µm column reduced run time by 50 % and solvent usage by 27.5 %, while the 4 µm superficially porous column achieved 70 % time and 62.5 % solvent savings.

Benefits and Practical Applications

This modernization approach allows pharmaceutical laboratories to:

• Accelerate batch release testing and routine QC analyses.
• Lower operational costs through reduced solvent use.
• Leverage scalable column chemistries for seamless method transfer without full revalidation.

Future Trends and Potential Applications

Opportunities to further enhance pharmacopeial assays include:

• Adoption of narrower bore columns (e.g., 3.0 mm id) to further cut solvent consumption.
• Integration of sub-2 µm particle UHPLC for ultra-fast separations where instrument pressure limits allow.
• Use of greener solvent systems and automated method scouting via digital platforms.

Conclusion

By following revised USP <621> guidelines, the acetaminophen and caffeine tablets HPLC assay was successfully modernized to smaller particle columns without revalidation. The updated method achieved equivalent or superior chromatographic performance, substantially reduced analysis time, and minimized solvent consumption, making it an attractive strategy for contemporary pharmaceutical QC laboratories.

References

• USP Harmonized Standards Home Page. Supplement USP Stage 4 Harmonization, Official, December 1, 2022.
• USP Monographs Acetaminophen and Caffeine Tablets, United States Pharmacopeia: 2024.
• Fu, R.; Grover, M.; Freeman, R.; Long, W. Understanding the Latest Revisions to USP <621>. Agilent Technologies white paper, 5994-6618EN, 2023.
• Fu, R. Modernizing the USP Ceftizoxime Sodium HPLC Method Following the Revised USP <621> Guidelines. Agilent Technologies application note, 5994-7819EN, 2024.