Instrument Considerations for Successful Adaptation of Amino Acid Analysis Methods Which Utilize Pre-Column Derivatization From an ACQUITY UPLC to an ACQUITY UPLC H-Class PLUS Binary System

Significance of the Topic

Amino acid analysis by liquid chromatography presents unique challenges due to the absence of native chromophores and diverse analyte chemistries. Pre-column derivatization, such as AccQ•Tag Ultra, enables UV detection and consistent retention behavior across a broad range of amino acids.

Objectives and Study Overview

This application note describes the transfer of established AccQ•Tag derivatization methods from the original ACQUITY UPLC system to the newer ACQUITY UPLC H-Class PLUS Binary System while retaining critical performance criteria including peak shape, resolution, sensitivity, linearity, and precision across multiple application areas.

Methodology

• Preparation of calibration standards (1–500 µM for most amino acids, 0.5–250 µM for cysteine) with norvaline as internal standard.
• Derivatization following Waters AccQ•Tag Ultra protocol.
• Optimization of injection and chromatographic parameters: use of a 30 µL flow-through needle, 95:5 water:acetonitrile needle-wash, and column temperature set to 45 °C.

Used Instrumentation

• ACQUITY UPLC H-Class PLUS Binary System with active solvent pre-heating and flow-through needle injector.
• AccQ•Tag Ultra column, reagents, and eluents from Waters.
• Empower 3 chromatography data system.

Main Results and Discussion

• Switching to a larger needle improved histidine peak symmetry (asymmetry increased from 0.50 to 0.66).
• Implementing a 95:5 water:acetonitrile needle wash minimized strong-solvent effects without carryover.
• Reducing column temperature to 45 °C balanced early-eluter peak shape and maintained cysteine–lysine resolution.
• Calibration linearity demonstrated R² > 0.999 with deviations < 5% at LOQ.
• Intra- and interday precision for retention time and quantitation showed RSDs < 0.3% over three days.
• Adaptation to cell culture, food/feed, and alkylated cysteines standards achieved satisfactory resolution (> 1.4) and symmetry.
• Quantitative taurine analysis in energy drinks matched within 1.6% between the two systems.

Benefits and Practical Applications

• Seamless migration of legacy methods to modern UPLC hardware without compromising analytical performance.
• Enhanced gradient precision and throughput support high-throughput workflows.
• Flexibility for global laboratories to run consistent amino acid analyses across different instrument generations.

Future Trends and Opportunities

Ongoing advancements in UPLC instrumentation will foster automated method transfer protocols, expanded derivatization chemistries, and digital-first workflows employing predictive modelling to streamline amino acid profiling in new matrices and emerging industries.

Conclusion

The AccQ•Tag Ultra amino acid analysis methods were successfully adapted to the ACQUITY UPLC H-Class PLUS Binary System, preserving key figures of merit and ensuring robust quantitative and qualitative performance across diverse sample types.

References

1. Waters Corporation. Amino Acid Standard Kits: Care and Use Manual; 2020.
2. Waters Corporation. Amino Acid Analysis Application Notebook; 2018.
3. Cohen S. Analysis of Sulfur-Containing Amino Acids III: Alkylation of Cysteine. Waters Lab Highlights; 1988.
4. Hong P, Wheat TE, Mazzeo JR, Diehl DM. Monitoring Cell Culture Media with the Waters Amino Acid Analysis Solution; 2007.