Method transfer of a USP-derived acetaminophen assay from an UltiMate 3000 SD system to a Vanquish Flex UHPLC system

Importance of the Topic

The reliable transfer of chromatographic assays between HPLC platforms is essential for pharmaceutical quality control, minimizing revalidation efforts and ensuring consistent results across laboratories and instruments.

Objectives and Study Overview

This study demonstrates the seamless migration of a United States Pharmacopeia (USP)–derived acetaminophen assay, including its impurities, from a Thermo Scientific™ UltiMate™ 3000 SD HPLC system to a Thermo Scientific™ Vanquish™ Flex UHPLC system. Key goals were to maintain chromatographic performance without altering core method parameters.

Methodology and Instrumentation

Reagents included LC/MS-grade methanol, phosphate buffers, acetaminophen, 4-aminophenol, and related impurity standards. Stock solutions were prepared in methanol and diluted to yield 1 mg/mL API and 10 μg/mL impurities. Separation employed a Hypersil GOLD™ C8 column (4.6 × 100 mm, 3 μm) at 35 °C, with a phosphate buffer/methanol gradient (1%–81% B) at 1 mL/min. Detection was performed at 230 nm with a 0.5 s response time and 1 μL injections. Unique features of the Vanquish Flex platform—adjustable autosampler idle volume and switchable column thermostatting and eluent pre-heating—were leveraged to replicate the gradient delay volume of the origin system.

Instrumentation

• UltiMate 3000 SD Quaternary HPLC: LPG-3400SD pump; WPS-3000TSL autosampler; TCC-3000SD column compartment (±7 μL pre-heater); DAD-3000 detector, 13 μL flow cell.
• Vanquish Flex UHPLC: Quaternary Pump F; Split Sampler FT; Column Compartment H; FG diode array detector, 13 μL flow cell.

Main Results and Discussion

Initial transfers showed earlier elution on Vanquish Flex due to lower gradient delay volume (GDV) and minor thermostat differences. By increasing the autosampler idle volume from 25 μL to 53 μL (without pre-heating) and to 79 μL (with pre-heating), retention time deviations for gradient peaks were reduced below 0.3%. System suitability criteria were met or exceeded: critical peak resolution >3.2, tailing factors 0.99–1.1, and peak height RSD <0.5%. Signal-to-noise ratios improved on the Vanquish Flex system, while relative impurity areas remained consistent.

Benefits and Practical Applications

• Maintains USP assay integrity without modifying core parameters.
• Reduces or eliminates revalidation effort by fine-tuning GDV via idle volume adjustment.
• Improves sensitivity and signal-to-noise performance on UHPLC platforms.

Future Trends and Applications

Adjustable volume control and flexible thermostatting modes will facilitate broader method portability across diverse LC platforms. As UHPLC pressures increase, active management of frictional heating and further miniaturization of dwell volumes will support high-throughput and high-resolution analyses. Continued development of standardized transfer guidelines and software-assisted volume matching will enhance interoperability among chromatographic systems.

Conclusion

The transfer of a USP acetaminophen assay from an UltiMate 3000 SD system to a Vanquish Flex UHPLC system was achieved successfully by exploiting adjustable autosampler idle volume and column thermostatting features. Equivalent chromatographic performance was maintained, with improved signal-to-noise and system suitability metrics, demonstrating a robust approach for regulated method migration.

References

1. Swartz M. E.; Krull I. Analytical Method Transfer; LCGC North America 2006, 24(11), 1204–1214.
2. Ermer J.; Limberger M.; Lis K.; Wätzig H. The transfer of analytical procedures; J. Pharm. Biomed. Anal. 2013, 85, 262–276.
3. Paul C.; Grübner M.; Steiner F. An instrument parameter guide for successful (U)HPLC method transfer. Thermo Fisher Scientific White Paper 72711, 2018.
4. United States Pharmacopeia (USP 40–NF 35). Acetaminophen assay. The United States Pharmacopeial Convention, 2017.
5. Grübner M.; Paul C.; Steiner F. Transfer of a USP derived acetaminophen assay from an Agilent 1260 system to an UltiMate 3000 SD system and a Vanquish Flex system. Thermo Fisher Scientific Application Note 72717, 2018.
6. United States Pharmacopeia (USP 40–NF 35) General Chapter <621> Chromatography. The United States Pharmacopeial Convention, 2017.