Quantitation of Amino Acids in Soy Flour, Dried Cow's Milk Powder, and Corn Silage by Triple Quadrupole LC/MS/MS

Importance of the topic

Amino acids are key nutritional and functional markers in food and feed analysis. Reliable quantitation supports quality control, regulatory compliance, and nutritional labeling in agricultural and dairy industries. Advanced analytical methods that avoid derivatization and deliver high throughput and sensitivity are in growing demand.

Goals and overview of the study

This study aimed to develop and validate a fast, robust UHPLC-MS/MS method for detecting and quantifying underivatized amino acids in complex matrices. Application targets included soy flour (NIST 3234), dried cow’s milk powder, and corn silage. The method was benchmarked against reference values and evaluated for specificity, linearity, accuracy, and matrix effects.

Methodology and instrumentation

The analytical workflow combined tailored hydrolysis protocols, HILIC separation, and triple-quadrupole detection.

• Sample preparation: protein hydrolysis with performic acid oxidation for cystine and methionine, 6 N HCl digestion for most amino acids, and 4 N NaOH for tryptophan.
• Chromatography: Agilent InfinityLab Poroshell 120 HILIC-Z column (2.7 µm, 2.1 × 150 mm) at 25 °C with a 14 min gradient (10 mM ammonium formate/0.1% formic acid in water and 90% ACN).
• Mass spectrometry: Agilent Jet Stream ESI in positive/negative switching with dynamic MRM transitions, optimized fragmentor voltages and collision energies.

Used Instrumentation

• Agilent 1290 Infinity II multislots autosampler, pump, column thermostat
• Agilent 6470 triple quadrupole LC/MS with Jet Stream ESI source
• Agilent MassHunter Acquisition and Quantitative Analysis software

Main results and discussion

The method achieved baseline separation of leucine/isoleucine isomers and full resolution of 20 amino acids. Calibration curves over 1–2,500 ng/mL showed R² > 0.99. Reagent blank spike recoveries ranged from 90 to 115% (methionine 81.5%). Post-extraction matrix-matched recoveries in feed and powder samples fell within 70–130%. Quantified values in NIST soy flour deviated by –2.4 to 18.3% from certified values, demonstrating high accuracy.

Benefits and practical applications

This approach eliminates time-consuming derivatization and ion-pairing reagents while maintaining high sensitivity and selectivity. It supports routine QA/QC and nutritional profiling in feed and dairy laboratories. Fast run times and robust performance enable high throughput screening.

Future trends and potential applications

• Integration of stable isotope internal standards for absolute quantitation.
• Automation of hydrolysis steps for increased reproducibility.
• Expansion to broader metabolomic panels using HILIC-MS/MS.
• Adoption in regulatory methods and interlaboratory proficiency testing.

Conclusion

A streamlined UHPLC-MS/MS workflow for underivatized amino acids in agricultural matrices was developed and validated. With excellent specificity, linearity, accuracy, and throughput, this method offers a practical solution for routine amino acid analysis in food and feed quality control.

Reference

• Otter D. E. Standardized Methods for Amino Acid Analysis of Food. Br J Nutr 2012, 108, 5230–5237.
• AOAC Official Method 994.12 Amino Acids in Feeds. 2000.
• Hsiao J. J. et al. The Use of HILIC Zwitterionic Phase Superficially Porous Particles for Metabolomics Analysis. LCGC Special Issues 2018, 36, 30–35.
• Zhao H.; Zulkoski J.; Mastovska K. Development and Validation of a Multiclass, Multiresidue Method for Veterinary Drug Analysis in Infant Formula and Related Ingredients Using UHPLC-MS/MS. J Agric Food Chem 2017, 65, 7268–7287.