Development and Validation of Non-derivatization LC/MS/MS Method for Fast Determination of Proteinogenic Amino Acids in Fish

Significance of the Topic

Amino acids are essential nutrients and key indicators of protein quality in fish. Rapid and accurate quantitation supports food quality control, nutritional evaluation, and research applications. Conventional HPLC methods with derivatization are laborious and time-consuming, driving the need for streamlined analytical workflows.

Objectives and Study Overview

This study aimed to develop and validate a non-derivatization LC-MS/MS method using MRM for fast quantitation of 20 proteinogenic amino acids and taurine in hydrolyzed fish samples. Performance was compared with a conventional UHPLC-fluorescence approach.

Methodology and Instrumentation

Samples of red snapper, seabass, and Spanish mackerel were hydrolyzed with 6N HCl/propionic acid (1:1) at 160 °C. Hydrolysis time was optimized between 15 and 120 min, with 30 min selected for best yields. Defatting was performed with hexane, followed by dilution and 0.2 µm filtration. Instrumentation included a Shimadzu LCMS-8060 triple quadrupole coupled to a Nexera UHPLC system with an Imtakt mixed-mode column (100 × 3 mm, 3 µm). MRM detection (and SIM for glycine) enabled direct analysis without derivatization, while a UHPLC-RF detector provided comparative data.

Main Results and Discussion

Calibration curves demonstrated linearity (R² > 0.995) over 0.01–20 µM for most analytes (glycine 5–20 µM). LOQs ranged from 0.004 to 0.10 µM. Intra-day precision (%RSD) was <8% for all amino acids. Hydrolysis optimization showed maximum recovery at 30 min. Matrix effects were assessed via post-spiking and corrected in quantitation. Analysis of three fish species yielded total essential amino acids of 8.5–11.9 g/100 g. Tryptophan losses under acid conditions were noted, indicating the need for alkaline hydrolysis for its determination. Asparagine and glutamine were converted to aspartic and glutamic acids, respectively.

Benefits and Practical Applications

• Accelerated turnaround with sample prep and analysis under 40 min.
• Elimination of derivatization simplifies workflow and reduces reagent costs.
• High specificity and sensitivity suitable for routine food QA/QC and nutritional studies.

Future Trends and Applications

• Application to diverse food and biological matrices.
• Incorporation of alkaline hydrolysis protocols to include reliable tryptophan analysis.
• Automation and high-throughput integration for comprehensive metabolomic profiling.

Conclusion

The validated non-derivatization LC-MS/MS method with rapid high-temperature acid hydrolysis offers a fast, accurate, and reliable alternative to traditional amino acid analysis in fish, matching fluorescence-based results while enhancing laboratory efficiency.

References

• European Pharmacopoeia 5.0. 2.2.56 Amino Acid Analysis. 2005.
• Bimal Mohanty et al. Amino Acid Compositions of 27 Food Fishes and their Importance in Clinical Nutrition. Journal of Amino Acids. 2014; Article ID 269797.
• Imtakt Technical Report T1734E.
• Matsumoto K., Watanabe J., Yazawa I. ASMS 2014, TP 510.