Method transfer of a USP derived acetaminophen assay from an Agilent 1260 Infinity system to an UltiMate 3000 SD system and a Vanquish Flex UHPLC system

Importance of the topic

The reliable transfer of liquid chromatographic methods between instruments is critical in pharmaceutical analysis, quality control, and regulated environments. Maintaining identical retention times, resolution, and sensitivity avoids costly revalidation and ensures consistent results across laboratories. Modern HPLC platforms with flexible gradient delay volumes and adjustable thermal controls facilitate smooth method transfer, reducing downtime and enhancing productivity.

Objectives and study overview

This study demonstrates the transfer of a USP-derived assay for acetaminophen and its impurities from an Agilent 1260 Infinity quaternary system to two Thermo Fisher Scientific platforms: the UltiMate 3000 Standard-D quaternary system and the Vanquish Flex UHPLC quaternary system. The goal is to achieve equivalent chromatographic performance without altering the original method parameters by leveraging hardware adaptations.

Methodology and instrumentation

Chromatographic conditions were kept constant: Hypersil GOLD C8 column (4.6×100 mm, 3 µm), phosphate buffer/methanol gradient, 1 mL/min flow, 35 °C column temperature, 230 nm detection, 1 µL injection. Sample contains 1 mg/mL acetaminophen and 10 µg/mL of each impurity.

Instrumentation summary

• Origin system: Agilent 1260 Infinity quaternary pump, thermostatted autosampler, diode array detector.
• Target systems: UltiMate 3000 SD quaternary pump with optional 7 µL eluent pre-heater and interchangeable static mixers; Vanquish Flex quaternary pump with active pre-heater, exchangeable sample loops, adjustable autosampler idle volume, and LightPipe™ DAD options.

Main results and discussion

On the UltiMate 3000 SD, installing a 7 µL pre-heater and increasing static mixer volume from 350 µL to 750 µL reduced gradient delay volume (GDV) differences. A gradient pre-start offset of –0.27 min aligned retention times within 1 % of the original. For the Vanquish Flex, replacing the default 25 µL loop with 100 µL and raising idle volume to 68 µL achieved comparable retention (≤1.7 % deviation) without a gradient pre-start. Column thermostat mode (still vs forced air) had minimal impact under these conditions. Peak resolution, asymmetry, and precision (RSD < 1 %) were maintained across all systems. Signal-to-noise ratios improved on both Thermo systems, and using the LightPipe DAD HL with a 60 mm flow cell delivered the highest sensitivity.

Benefits and practical applications

• Hardware adjustments (pre-heaters, mixers, sample loops, idle volume) enable transfer without method revalidation.
• Enhanced flexibility supports migration from legacy to UHPLC systems.
• Improved sensitivity via LightPipe technology benefits trace impurity analysis.

Future trends and potential applications

The development of adaptive system architectures with real-time GDV calibration may further streamline transfers. Integration of machine-learning algorithms for automated parameter optimization and the increasing use of high-sensitivity detectors will expand capabilities for challenging trace analyses and ultra-high-throughput environments.

Conclusion

Through strategic use of eluent pre-heaters, variable static mixers, sample loops, and adjustable idle volumes, the acetaminophen assay was successfully transferred from an Agilent 1260 Infinity to UltiMate 3000 SD and Vanquish Flex systems. Equivalent chromatographic performance and enhanced sensitivity were achieved without altering the validated method, underscoring the value of flexible hardware solutions in method migration.

Reference

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2. Ermer J et al. The transfer of analytical procedures; J Pharm Biomed Anal 2013, 85:262–276.
3. Paul C, Grübner M et al. Thermo Scientific White Paper 72711: An instrument parameter guide for successful (U)HPLC method transfer, 2018.
4. United States Pharmacopeial Convention. USP40-NF35 S2, Acetaminophen method, 2017.
5. USP40-NF35 S2, General chapter <621> Chromatography, 2017.
6. Manka A, Franz H. Thermo Scientific Technical Note 165: Boosting Trace Detection Performance with the Vanquish DAD and LightPipe, 2016.