Amino Acid Analysis of Spinach and Apple by UHPLC

Importance of the Topic

Amino acid profiling is essential for nutritional assessment, quality control and research in food and plant sciences. Automated online precolumn derivatization coupled with UHPLC offers high sensitivity, selectivity and throughput, enabling rapid analysis of complex biological matrices.

Objectives and Study Overview

This work aimed to transfer a conventional HPLC amino acid analysis method using Eclipse Plus C18 columns to a UHPLC platform (Agilent 1290 Infinity), evaluate performance for spinach and apple matrices, test a QuEChERS extraction workflow, and assess method flexibility for protein hydrolysates.

Methodology and Instrumentation

Online Precolumn Derivatization:

• Primary amino groups derivatized with ortho-phthalaldehyde (OPA) and 3-mercaptopropionic acid at pH 10; UV detection at 338 nm.
• Secondary amino groups derivatized with 9-fluorenylmethyl chloroformate (FMOC) at pH 10; UV detection at 262 nm.

Chromatography:

• Agilent 1200SL with Eclipse Plus C18 (4.6×250 mm, 5 µm) and RRHT C18 (4.6×150 mm, 3.5 µm) columns; gradient run times 24–40 min.
• Agilent 1290 Infinity UHPLC with RRHT Eclipse Plus C18 (2.1×100 mm, 1.8 µm); run time 12 min at pressures up to 474 bar.
• Scaled injection programming: sample loop reduced from 100 µL to 20 µL with proportional draw and mix steps.

Instrumentation:

• UHPLC systems: Agilent 1200SL and 1290 Infinity.
• UV detectors with 10 mm Maxlight flow cell, slit width 4 nm.
• QuEChERS extraction using SampliQ AOAC salt and dispersive-SPE kits, followed by UHPLC/UV or GC/MS.

Key Results and Discussion

Transfer to UHPLC achieved improved chromatographic resolution (Rs > 2.3), linearity (R2 ≥ 0.999), limits of quantitation of 3–10 pmol/µL, and analysis time halved compared to HPLC. QuEChERS extraction allowed clear separation of 27 amino acids from apple and spinach, revealing distinct profiles: apples rich in aspartic acid, asparagine, alanine and GABA; spinach high in aspartic acid, glutamic acid, asparagine and arginine. Repeatability on the 1.8 µm column showed %RSD < 2% for major amino acids. Neutralization of acidic protein hydrolysates improved recovery to 95–110%.

Benefits and Practical Applications

• High-throughput amino acid profiling of food and plant samples.
• Enhanced sensitivity and selectivity for primary and secondary amines.
• Adaptable sample preparation via QuEChERS for diverse matrices.
• Reduced solvent use and faster analysis with UHPLC.

Future Trends and Opportunities

Integration with high-resolution mass spectrometry for comprehensive metabolite profiling; expansion to non-protein amino acids and bioactive peptides; automated sample preparation platforms; applications in plant metabolomics, functional food analysis and clinical nutrition studies.

Conclusion

The study demonstrates a robust transfer of an amino acid analysis method from HPLC to UHPLC, delivering faster, more sensitive and reproducible results. QuEChERS extraction is effective for fruit and vegetable matrices, and method flexibility supports customization for challenging samples, facilitating routine nutritional and quality assessments.

References

• Park S., Kim Y., Xu H., Boo H., Lee S. Amino Acid and GABA content in different cultivars of Momordica charantia L. Journal of Medicinal Plants Research. 2009;3(11):897–900.
• AOAC Method 2007.01: Amino Acids in Foods and Feeds by UHPLC following QuEChERS Extraction.