Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

Importance of the Topic

Accurate quantification of amino acids in food matrices is essential for nutritional assessment, quality control, and food safety evaluation. Conventional methods often require derivatization or ion-pairing reagents to retain highly polar amino acids on reversed-phase columns, increasing sample preparation time and risk of reagent interferences. A streamlined, derivatization-free LC-MS/MS approach addresses these challenges, offering rapid, sensitive, and selective detection of multiple amino acids in a single run.

Study Objectives and Overview

The primary goal of this work was to develop and validate a simultaneous high-sensitivity LC-MS/MS method for 20 proteinogenic amino acids in diverse food samples without chemical derivatization. The study explored mixed-mode stationary phases compatible with a volatile mobile phase to achieve adequate retention and separation, followed by application to real beverage and culinary samples.

Methodology

Sample Preparation:

• Standard solutions of 20 amino acids prepared in dilute formic acid or HCl.
• Food samples (sports beverages, sake, beer, sweet cooking rice wine) diluted 10–100-fold in 0.1 N HCl and filtered through 0.2 µm membranes.

Chromatographic Conditions:

• Column: Intrada Amino Acid, 3.0 mm I.D. × 50 mm, 3 µm particle size (Imtakt).
• Mobile Phase Case 1: A = acetonitrile/0.1% formic acid; B = 100 mM ammonium formate. Gradient from 14% to 100% B (0–10 min), return to 14% (10.01–15 min).
• Mobile Phase Case 2 (high-resolution): A = acetonitrile/tetrahydrofuran/25 mM ammonium formate/0.3% formic acid; B = 100 mM ammonium formate/acetonitrile (80/20). Optimized gradient for enhanced resolution.
• Flow rate: 0.6 mL/min; column temperature: 40 °C; injection volume: 2 µL.

Used Instrumentation

• LC System: Shimadzu Nexera UHPLC.
• Mass Spectrometer: Shimadzu LCMS-8050 triple quadrupole with ESI source in positive MRM mode.
• Key features: UF-MRM at 555 ch/sec, high-speed polarity switching (5 ms).

Main Results and Discussion

Optimization of MRM transitions and ESI heating gas temperature enabled sensitive detection of all 20 amino acids without derivatization. Case 1 conditions provided sufficient retention and peak shape for quantitative work; Case 2 improved separation but increased mobile phase complexity. Analytical performance under Case 1:

• Linearity ranges: 0.01–500 nmol/mL with correlation coefficients (r2) ≥ 0.99.
• Repeatability: RSD values mostly below 5%; a few amino acids up to 11% at low concentrations.

Application to commercial sports beverages, Japanese sake, beer, and mirin demonstrated successful detection of labeled amino acids and endogenous profiles with minimal sample processing.

Benefits and Practical Applications

• Eliminates derivatization and ion-pair reagents, reducing sample prep time and potential contaminants.
• High throughput: single-run analysis of 20 amino acids in under 15 minutes.
• Wide dynamic range suitable for trace to major amino acids in complex food matrices.
• Applicable to routine QA/QC, nutritional labeling, and food authenticity studies.

Future Trends and Opportunities

Further advancements may include integration of stable isotope-labeled internal standards for absolute quantitation, expansion to modified or non-proteinogenic amino acids, and coupling with automated solid-phase extraction. Emerging high-resolution MS platforms and online sample cleanup promise real-time monitoring in food processing and metabolomics workflows. Miniaturization and microfluidic interfaces could enable on-site food testing.

Conclusion

A robust, derivatization-free LC-MS/MS method using a mixed-mode column and volatile mobile phases was established for rapid, sensitive quantification of 20 amino acids in food samples. The approach simplifies sample preparation, maintains excellent analytical performance, and is readily applicable to various food and beverage matrices.

Reference

• Matsumoto K, Watanabe J, Yazawa I. Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS. Shimadzu Corporation Application Note PO-CON1453E; June 2014.