Retention Time Tables for Amino Acids Using the AccQ•Tag Method

Importance of Topic

Amino acid profiling is fundamental in biochemical research, food quality control, clinical diagnostics and pharmaceutical development. Robust separation and precise retention time data ensure reliable identification of both proteinogenic and non-proteinogenic amino acids. Understanding retention properties under varied conditions supports method optimization and the discovery of novel biomarkers.

Objectives and Overview

This application note evaluates retention coefficients of over 60 amine-containing compounds using the AccQ•Tag derivatization approach. Two separation protocols at eluent pH 5.05 and pH 5.80 are compared to extend standard protein hydrolysate analyses to modified, rare or non-hydrolysate amines. Retention coefficients (RC) are calculated relative to phenylalanine to facilitate prediction of elution order in diverse sample matrices.

Methodology and Instrumentation

The AccQ•Tag method uses 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for rapid derivatization of primary and secondary amines. Key parameters include:

• Derivatization reagent: AQC
• Chromatographic system: Waters HPLC with AccQ•Tag column
• Eluent conditions: protocol A at pH 5.05 (45 min gradient), protocol B at pH 5.80 (60 min gradient)
• Temperature and ionic strength optimized per protocol

Main Results and Discussion

Tables of retention coefficients reveal trends in elution behavior:

• At pH 5.05, retention coefficients range from 0.186 for beta-hydroxyaspartic acid to 1.034 for tryptophan.
• At pH 5.80, the gradient extension improves resolution of Asn, Gln, hydroxyproline and modified derivatives; RC values shift and some peak orders reverse (e.g., Ser/Glu, Arg/Thr, Cys/Tyr).
• Differences in RC greater than 0.015 generally yield baseline resolution.

Sample type codes map compounds to matrices such as hydrolysates (1), glycoproteins (2), modified proteins (3), collagen (4), physiological samples (6) and novel intermediates (7).

Benefits and Practical Applications

The extended AccQ•Tag protocol allows laboratories to:

• Identify and quantify rare or modified amino acids not found in standard hydrolysates.
• Adjust separation parameters to resolve critical isobaric pairs.
• Apply a consistent derivatization and separation workflow across diverse sample types including biologics, clinical specimens and collagen digests.

Future Trends and Opportunities

Advances may include automated method development tools to predict retention of novel derivatives, integration with high-resolution mass spectrometry for confirmation, and expansion of compound libraries for metabolomics. Further optimization of gradient profiles and buffer compositions could enhance throughput and sensitivity.

Conclusion

The AccQ•Tag method with pH 5.05 and 5.80 protocols successfully extends amino acid analysis to a wide variety of amine compounds. Retention coefficient tables serve as a practical guide for anticipating elution behavior and optimizing separations in complex matrices.

Reference

1. Sensitive Analysis of Cystine/Cysteine using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatives, Techniques in Protein Chemistry IV, Academic Press, 1993.
2. Waters AccQ•Tag Amino Acid Analysis System Operator’s Manual Number 154-02TP, Waters Corporation, 1996.