Simultaneous Analysis of Amino Acids Using Automatic Pretreatment Function of Prominence-i Integrated LC System

Importance of the Topic

Amino acid analysis is essential for quality control and research in food, pharmaceutical and biochemical fields. Accurate profiling of proteinogenic and functional amino acids supports nutritional labeling, metabolic studies and process monitoring.

Objectives and Study Overview

This study demonstrates a fully automated pre-column derivatization workflow for simultaneous analysis of 23 amino acids using the Prominence-i LC-2030C integrated HPLC system. The aim is to streamline reagent mixing, reduce sample volume and maintain high sensitivity and reproducibility.

Methodology

Derivatization reagents include o-phthalaldehyde (OPA), 3-mercaptopropionic acid and 9-fluorenylmethoxycarbonyl chloride (FMOC). The system’s co-injection function aspirates sample and reagents in sequence into a single needle for in-needle mixing. Derivatized amino acids are separated on a low-pressure gradient using three mobile phases: acetate buffer (pH 6), water/acetonitrile (1:9) and acetate buffer (pH 5) with EDTA. Phosphoric acid solution is added post-derivatization to adjust pH.

Used Instrumentation

• Shimadzu Prominence-i LC-2030C integrated HPLC system
• Shim-pack XR-ODSII column (100 mm × 3.0 mm I.D., 2.2 µm)
• Fluorescence detector with dual channels (Ex/Em 350/450 nm and 266/305 nm)

Main Results and Discussion

Chromatographic separation of 20 proteinogenic amino acids achieved baseline resolution within 14 minutes. Additional separation of functional amino acids (citrulline, taurine, theanine, GABA) extended the profile to 23 analytes. Calibration showed linearity coefficients (r2) above 0.999 and area repeatability RSDs below 1% for all targets. Analysis of a diluted beer sample confirmed clear peak identification and quantitation under the same conditions.

Benefits and Practical Applications

• Automated in-needle derivatization minimizes reagent and sample consumption.
• Eliminates manual mixing vials, reducing preparation steps and contamination risks.
• High sensitivity from full injection of derivatized sample into column.
• Applicable to routine quality control in food, beverage and pharmaceutical laboratories.

Future Trends and Potential Applications

Integration of automated derivatization with UHPLC and mass spectrometry detection could further accelerate analysis and expand coverage to modified or non-proteinogenic amino acids. Software-driven workflows and on-board reagent storage will enhance throughput and robustness for high-volume laboratories.

Conclusion

The Prominence-i automated pre-column derivatization approach delivers rapid, reliable and sensitive analysis of 23 amino acids with minimal manual intervention. This method offers a practical solution for laboratories seeking to improve efficiency and data quality in amino acid profiling.