Analysis of Amino Acids in Animal Feed Matrices Using the Ultivo Triple Quadrupole LC/MS System

Significance of the Topic

Amino acids are essential building blocks for proteins and critical nutrients in animal and pet feed. Ensuring accurate quantitation of both essential and nonessential amino acids supports feed quality and animal health. Traditional sample preparation involving hydrolysis and derivatization is labor intensive, which limits throughput. Modern hydrophilic interaction chromatography (HILIC) coupled with high sensitivity LC/MS offers an underivatized workflow, reducing analysis time and complexity.

Objectives and Study Overview

This study evaluates a rapid and straightforward method for quantifying 17 underivatized amino acids in animal feed matrices using the Agilent Ultivo triple quadrupole LC/MS system in combination with HILIC separation. Key performance metrics including lower limits of quantitation (LLOQ), chromatographic precision, linear dynamic range, and accuracy are assessed to demonstrate method suitability for routine feed analysis.

Methodology

• Sample preparation consists of acid hydrolysis of ground animal feed with 6N HCl and phenol under nitrogen, followed by dilution and filtration without derivatization.
• Chromatographic separation is performed on a Poroshell 120 HILIC-Z column at 25 °C using a gradient of ammonium acetate/formic acid in water and acetonitrile.
• Detection employs positive ion mode dynamic multiple reaction monitoring (dMRM) on the Ultivo system.
• MRM transitions and MS parameters are optimized using MassHunter Acquisition and Quantitative Analysis software.

Instrumentation

• Agilent 1260 Infinity II Bio-Inert HPLC, including pump, multisampler, and multicolumn thermostat.
• Agilent Ultivo triple quadrupole LC/MS with Jet Stream electrospray ionization source.

Results and Discussion

Retention and baseline separation of all 17 amino acids, including critical isobaric pairs leucine and isoleucine, are achieved within a seven-minute window with retention time precision below 0.5% RSD. Calibration curves spanning low ppb to tens of ppm levels exhibit R2 values above 0.992. LLOQs range from 1.12 to 75 ppb depending on the analyte, with signal to noise ratios exceeding 3–10 at the LLOQ. Accuracy at the lowest calibration levels is within 11.5%, and precision across replicate injections in matrix remains below 1.1% RSD.

Benefits and Practical Applications

This method eliminates derivatization, simplifying the workflow and reducing potential sources of error. The compact Ultivo footprint and robust quantitation capability make it well suited for high-throughput feed testing laboratories requiring reliable amino acid profiling for quality control and nutritional verification.

Future Trends and Possibilities

Advances in LC/MS system miniaturization and novel ion guide and collision cell technologies will further enhance laboratory efficiency and reduce maintenance. Integration of automated sample preparation and data analysis workflows will support expanded applications in feed, food, and biotechnology sectors. Emerging bioinformatic tools may enable deeper insights into amino acid profiles and nutritional optimization.

Conclusion

The described HILIC LC/MS approach on the Ultivo triple quadrupole system provides a fast, accurate, and sensitive solution for underivatized amino acid analysis in animal feed. The simplified sample preparation and excellent analytical performance support its adoption for routine feed quality assessment and regulatory compliance.

References

• Methods for the Analysis of Underivatized Amino Acids by LC/MS, Agilent Technologies Application Note, publication number 5991-8582EN.
• Quantitative Analysis of Underivatized Amino Acids in Plant Matrix by Hydrophilic Interaction Chromatography with LC/MS Detection, Agilent Technologies Application Note, publication number 5991-8922EN.
• Analysis of Underivatized Amino Acids by LC/MS for Bioreactor Cell Culture Monitoring, Agilent Technologies Application Note, publication number 5991-8816EN.
• Otter DE. Standardised methods for amino acid analysis of food. British Journal of Nutrition. 2012;108:S230-S237.
• Wu G. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition. Journal of Animal Science and Biotechnology. 2014;5:34-45.