Amino Acid Profiling of Fish Feeds Using Agilent 1260 Infinity II LC with DAD and FLD

Importance of the Topic

Amino acid profiling of fish feeds provides essential insight into feed quality and nutritional adequacy. Accurate measurement of individual amino acids supports optimized diet formulation for fish growth and health, ensures balanced protein intake, and helps monitor functional additives such as taurine, theanine, glucosamine, and cysteine that enhance immunity and performance in aquaculture.

Study Objectives and Overview

This study refined an existing chromatographic protocol to quantify 24 amino acids in two commercial fish feeds. The goals were to incorporate four additional targets (taurine, theanine, glucosamine, and cysteine) into a robust method, assess performance metrics, and demonstrate applicability to real feed samples following acidic hydrolysis.

Methodology and Instrumentation

A precolumn derivatization approach using o-phthalaldehyde (OPA) for primary amino acids and 9-fluorenylmethyl chloroformate (FMOC) for secondary amino acids was automated on the Agilent 1260 Infinity II Multisampler. Separation was achieved on an Agilent AdvanceBio AAA stationary phase under a gradient of phosphate/borate buffer and a water–methanol–acetonitrile blend. Detection combined a Diode Array Detector (DAD) operated at 338 nm and a Fluorescence Detector (FLD) with excitation at 345 nm and emission at 455 nm. Acidic hydrolysis (6 N HCl, 110 °C, 24 h) prepared feed samples for analysis.

Main Results and Discussion

All 24 amino acids were baseline resolved within a 24 min run. FLD offered superior sensitivity for most analytes, while DAD provided enhanced detection of cysteine and cystine due to their low fluorescence yield. Limits of detection and quantification were ≤0.45 and ≤0.90 pmol/µL for all amino acids except cysteine (4.5 and 9 pmol/µL). Linearity (R²>0.999), retention time precision (<0.4 % RSD), and area precision (<1 % RSD) confirmed method robustness. Recoveries in fish matrices ranged between 70 % and 130 %, indicating minimal matrix bias.

Benefits and Practical Applications

The combined DAD/FLD configuration delivers both selectivity and sensitivity across diverse amino acid chemistries. Automated derivatization reduces manual steps and improves reproducibility. This workflow supports quality control of commercial feeds, research into functional feed ingredients, and regulatory compliance in aquaculture nutrition.

Future Trends and Potential Applications

• Extension to non-proteinogenic and modified amino acids in specialty feeds.
• Integration with high-resolution mass spectrometry for structural confirmation.
• Miniaturized or high-throughput platforms to increase sample throughput.
• Application to other agricultural products such as plant protein isolates.

Conclusion

An optimized Agilent 1260 Infinity II LC method with DAD and FLD detectors enables reliable quantitation of 24 amino acids in fish feeds. The protocol demonstrates excellent sensitivity, precision, and recovery, making it a valuable tool for aquaculture feed evaluation and research.

Used Instrumentation

• Agilent 1260 Infinity II Binary Pump
• Agilent 1260 Infinity II Multisampler
• Agilent 1260 Infinity II Multicolumn Thermostat
• Agilent 1260 Infinity II Diode Array Detector (fixed slit)
• Agilent 1260 Infinity II Fluorescence Detector
• Agilent AdvanceBio AAA LC column and guard column

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