Separation of two sulfurated amino acids with other seventeen amino acids by HPLC with pre-column derivatization

Importance of the Topic

A robust HPLC method enabling simultaneous separation of 19 amino acids, including two sulfurated residues, is essential for accurate quality control in feed, food and pharmaceutical industries. Sulfurated amino acids are prone to degradation under common hydrolysis conditions and demand optimized derivatization and chromatographic conditions for reliable quantitation.

Objectives and Study Overview

This work describes the development of a simple and reliable reverse-phase HPLC method with pre-column OPA and FMOC derivatization to separate 19 amino acids, including methionine sulfone and cysteic acid. The method was first optimized on an Agilent 1200 SL RRLC and then successfully transferred to an Agilent 1100 quaternary HPLC system.

Methodology and Instrumentation

Derivatization was performed using OPA for primary amino acids and FMOC for proline. Two chromatographic setups were compared:

• Agilent 1200 SL RRLC with Zorbax Eclipse Plus C18 (4.6×50 mm, 1.8 µm), binary pump, DAD; separation in ~10 min, 14.1 min cycle.
• Agilent 1100 Series HPLC with Zorbax Eclipse Plus C18 (4.6×150 mm, 5 µm), quaternary pump, VWD; separation in ~28 min, 32.5 min cycle.

Mobile phase A was 10 mM phosphate-borate buffer (pH 8.2), mobile phase B was acetonitrile:methanol:water (45:45:10). A gradient elution was tailored to each system accounting for delay volumes.

Main Results and Discussion

Both systems achieved baseline separation of all 19 amino acid derivatives. The RRLC method provided over twofold faster analysis and higher sensitivity due to reduced peak broadening. Transferring the method to the 1100 HPLC required adjusting gradient times to compensate for larger system volume, ensuring stable retention and resolution of early eluting polar derivatives.

Benefits and Practical Applications

The method supports routine QA/QC of feed, food and pharmaceutical matrices containing sulfurated amino acids. Improved throughput and sensitivity facilitate high-volume testing and comprehensive product quality evaluation.

Future Trends and Applications

Further advancements may include coupling with UHPLC-MS/MS for expanded analyte panels, fully automated derivatization workflows for enhanced reproducibility, and application to complex biological matrices.

Conclusion

The developed pre-column derivatization HPLC method reliably separates 19 amino acids including challenging sulfurated compounds. It demonstrates easy transfer between RRLC and conventional HPLC platforms, doubling throughput while maintaining robust performance.

Reference

1. Henderson JW Jr, Brooks A. Agilent Application Note 5990-4547EN (2009)
2. Woodward C, Henderson JW Jr. Agilent Application Note 5989-6297EN (2007)