Evaluation of intra- and inter-system precision for HPLC analysis of active pharmaceutical ingredients and impurities

Importance of the topic

High-performance liquid chromatography (HPLC) remains a cornerstone in pharmaceutical analysis, providing essential support for research, development, and quality control. Demonstrating reproducible results across multiple HPLC systems allows laboratories to transfer methods seamlessly, ensures data integrity, and boosts confidence in analytical outcomes.

Objectives and study overview

• Assess hardware-related variability of six Thermo Scientific Vanquish Core HPLC systems operating under identical, highly controlled conditions.
• Evaluate intra-system and inter-system precision for retention time and peak area using two applications: nevirapine with its impurities and acetaminophen with related impurities.

Methodology and instrumentation

Samples and mobile phases were freshly prepared by a single operator and strictly controlled to minimize non-hardware-related variability. Two systems at a time were run in parallel, with columns, samples, and solvents rotated among systems. Data were acquired and processed using Thermo Scientific Chromeleon CDS 7.3.

• Instrument: Thermo Scientific Vanquish Core Quaternary HPLC systems equipped with a quaternary pump, cooled autosampler, column compartment, variable wavelength detector (standard flow cell), and nanoViper capillaries.
• Columns: Acclaim Polar Advantage II (150 x 3 mm, 3 µm) for nevirapine application; Hypersil GOLD C8 (100 x 4.6 mm, 3 µm) for acetaminophen application.
• Mobile phases: Ammonium acetate buffer/acetonitrile gradient (pH 5) for nevirapine; phosphate buffer/methanol gradient for acetaminophen, based on USP monographs.
• Sample preparation: Stock and working solutions of APIs and impurities prepared in appropriate solvents following USP procedures; injection volume 5 µL; detection at 230 nm or 240 nm.

Main results and discussion

Void volume equivalency was confirmed across all six systems (corrected system void volumes 42.1–43.1 µL), indicating negligible impact from capillaries or flow paths. Intra-system precision on each system yielded retention time RSDs of 0.01–0.12% and peak area RSDs of 0.05–0.31% for both applications. Inter-system comparison showed retention time RSDs below 1% and peak area RSDs below 2.5% for all selected analytes, supporting robust method transfer without adaptation of processing parameters.

Benefits and practical applications

• Ensures high reproducibility and consistency across laboratories using identical Vanquish Core systems.
• Facilitates efficient method transfer and validation in regulated environments.
• Reduces revalidation efforts and enhances confidence in QC results.

Future trends and opportunities

Analytical workflows will benefit from further automation, integration of advanced software for real-time data monitoring, and incorporation of artificial intelligence for predictive maintenance. Expanding multi-system comparability studies and applying similar approaches to LC–MS platforms will enhance overall laboratory efficiency.

Conclusion

This study demonstrates that six Thermo Scientific Vanquish Core HPLC systems provide excellent intra- and inter-system precision for pharmaceutical assays. Tight control of hardware variability ensures reliable method transfer and consistent analytical performance across multiple instruments.

References

1. Thermo Fisher Scientific Technical Note 73337. Global round robin test of thiopental EP method performance. 2019.
2. Thermo Fisher Scientific Technical Note 73915. Complete workflow for determining nevirapine and related impurities. 2021.
3. United States Pharmacopeia USP40-NF35 S2. Acetaminophen assay method. 2017.
4. United States Pharmacopeia USP40-NF35 S1. Nevirapine assay method. 2017.
5. Thermo Fisher Scientific Technical Note 73786. Long-term robustness of thiopental Ph. Eur. method performance. 2020.