Instrument Considerations for Successful Adaptation of Amino Acid Analysis Methods which Utilize Pre-Column Derivatization from an ACQUITY UPLC to an ACQUITY Premier Binary System

Importance of the Topic

The accurate and reproducible analysis of amino acids is essential across pharmaceutical, biopharmaceutical and food-testing laboratories. Most amino acids lack a native chromophore and exhibit diverse chemical properties, posing challenges in detection and separation. Pre-column derivatization with AccQ•Tag reagents and adoption of modern UPLC systems enhance sensitivity, throughput and method robustness.

Study Objectives and Overview

This application note describes the migration of a legacy amino acid analysis (AAA) method using AccQ•Tag derivatization from the ACQUITY UPLC system to the newer ACQUITY Premier Binary System. Key goals include maintaining critical performance metrics—peak shape, resolution, linearity, detection limits and precision—across four application areas: protein hydrolysates, cell culture media, food and feed matrices, and alkylated cysteine samples. A quantitative comparison of taurine in energy drinks further validates the adaptation.

Methodology and Instrumentation

Major differences between the two systems include injector design (fixed loop vs. flow-through needle), column heating (passive vs. active) and MaxPeak High Performance Surfaces technology. To preserve chromatographic performance on the ACQUITY Premier Binary System, the following adjustments were implemented:

• Use of 30 µL flow-through needle with 1 µL injection volume
• Needle wash solvent composition of 95:5 water:acetonitrile to minimize strong solvent effects
• Column temperature optimized to 45 °C for balanced peak symmetry and resolution
• Inline filter to reduce dispersion and protect column
• Empower 3 software for data acquisition and management

Main Results and Discussion

• Initial method transfer achieved separation of all 17 underivatized amino acids, but early-eluting histidine showed peak fronting due to solvent mismatch.
• Switching to a larger needle and low-organic wash solvent improved histidine asymmetry from 0.50 to 0.66 (USP tailing ≤ 0.9).
• Lowering column temperature from 55 °C to 45 °C further enhanced histidine symmetry (asymmetry 0.76) while maintaining cysteine–lysine resolution ≥ 1.7.
• Calibration linearity over 1–500 µM (0.5–250 µM for cysteine) yielded R2 > 0.999 and < 6% deviation at the limit of quantitation.
• Intra- and interday precision for retention time and concentration exhibited RSD ≤ 0.6% and ≤ 0.4%, respectively.
• Application to cell culture, food and feed standards, and alkylated cysteines achieved comparable resolution (≥ 1.2) and peak shapes without further method changes.
• Quantitative taurine analysis in two energy drink samples showed agreement within 1% between the two UPLC systems.

Benefits and Practical Applications

• Ensures consistent AAA performance when upgrading hardware or sharing methods across laboratories
• Maximizes recovery and reproducibility through MaxPeak surface technology
• Supports high-throughput workflows with precise, robust gradients on the Premier Binary System
• Enables reliable quantitation in diverse matrices including clinical, bioprocess and nutritional samples

Future Trends and Applications

Ongoing instrument innovations will focus on deeper integration of surface technologies, automated derivatization workflows and coupling to mass spectrometry for enhanced selectivity. Expansion of derivatization chemistries and microfluidic formats will further improve throughput, sensitivity and sustainability in amino acid analysis.

Conclusion

The AccQ•Tag Ultra AAA methods have been successfully migrated from the ACQUITY UPLC to the ACQUITY Premier Binary System without loss of chromatographic or quantitative performance. Key adjustments to injector size, wash solvent and column temperature preserved all critical metrics, ensuring method longevity and compatibility with new instrumentation.

Reference

• Waters Care and Use Manual for Amino Acid Standard Kits, 2020.
• Amino Acid Analysis Application Notebook, Waters, 2018.
• Cohen S. Analysis of Sulfur-Containing Amino Acids: Alkylation of Cysteine, 1988.
• Hong P., Wheat T.E., Mazzeo J.R., Diehl D.M. Monitoring Cell Culture Media with the Waters AAA Solution, 2007.