Agilent Approaches for Amino Acid Analysis

Importance of the Topic

Amino acid analysis remains a cornerstone technique in biochemistry, pharmaceutical development, food science and bioprocess monitoring. Quantitative and qualitative profiles of free and bound amino acids support protein characterization, cell culture media optimization, fermentation control and flavor profiling. Reliable methods are required to meet stringent quality, regulatory and research objectives across diverse industries.

Objectives and Overview

This article reviews a comprehensive suite of Agilent approaches to amino acid analysis, including historical methods and the latest innovations. Key topics include pre- and post-column derivatization with UV/fluorescence detection, HILIC LC/MS workflows for underivatized compounds, ion-pairing techniques and chiral separations. The evolution of column chemistries and automated reagent kits is described, alongside practical guidance on method implementation.

Methodology and Instrumentation

Derivatization strategies combine orthophthalaldehyde (OPA) and fluorenylmethyloxycarbonyl (FMOC) for precolumn tagging of primary and secondary amino acids. Automated protocols in Agilent autosamplers perform multi-step mixing and injection, reducing manual error. An alternative HILIC-MS method employs an AdvanceBio MS Spent Media column under high-organic conditions and volatile buffers to analyze underivatized amino acids in positive or negative ion mode. Ion-pairing and chiral analyses extend capabilities for nonstandard and enantiomeric species.

• UHPLC systems: 1290 Infinity II Multisampler; 1260 Infinity II Vialsampler
• Columns: AdvanceBio AAA Poroshell (2.7 μm); ZORBAX Eclipse Plus C18; AdvanceBio MS Spent Media HILIC; chiral CROWNPAK
• Detectors: Diode array (UV), Fluorescence (FLD), 6230 TOF LC/MS
• Reagent kits: Borate buffer, OPA, FMOC, DTDPA and amino acid standards

Main Results and Discussion

Precolumn derivatization methods delivered baseline separation of 23 amino acids in under 18 minutes, with retention time RSDs mostly below 1% and peak area variability under 3%. Poroshell particles proved resilient to elevated pH and temperature, offering high efficiency with reduced back pressure. HILIC-MS workflows achieved unambiguous detection of native amino acids, including isomeric leucine/isoleucine resolution above pharmacopeial requirements. The elimination of derivatization reagents simplified sample processing and enabled multiplexed metabolite profiling.

Benefits and Practical Applications

• High throughput: automated derivatization and fast gradients support hundreds of samples per week
• Robustness: Poroshell columns tolerate pH 3–11, minimizing column degradation
• Sensitivity: UV/FLD detection for derivatized analytes and low-picomole MS detection of native compounds
• Versatility: workflows for protein hydrolysates, cell culture media, fermentation broths, and flavor matrices
• Cost savings: reduced reagent handling, combined assays for amino acids and other metabolites

Future Trends and Opportunities

Emerging directions include multidimensional separations, ultra-high-pressure LC for sub-2 μm particles, integration with ion mobility–MS and ambient ionization techniques. Artificial-intelligence–driven method development may streamline gradient and buffer optimization. Novel stationary phases and microfluidic platforms promise further miniaturization and environmental sustainability.

Conclusion

Agilent’s portfolio for amino acid analysis offers a unified toolkit spanning UV/FLD derivatization, HILIC-MS profiling, ion-pair and chiral methodologies. Automated protocols, versatile column architectures and robust instrumentation deliver reproducible performance across diverse applications, supporting both routine quality control and advanced research needs.

Reference

• Powell M. Agilent AdvanceBio Amino Acid Analysis How-To Guide. Agilent Technologies; 2017. Pub no. 5991-7694EN.
• Powell M. HILIC LC/MS for Spent Media and Metabolite Profiling. Agilent Technologies; 2018. Pub no. 5991-8816EN.
• Powell M. User Guide for ZORBAX Eclipse AAA Columns. Agilent Technologies; 2000. Pub no. 5980-3088EN.
• Powell M. Improved Amino Acid Methods using Agilent ZORBAX Eclipse Plus C18. Agilent Technologies; 2010. Pub no. 5990-4547EN.