UPLC-MS Analysis of 20 Amino Acids Using the Kairos Amino Acid Kit for Biomedical Research

Importance of the Topic

Amino acid profiling serves as a cornerstone in biomedical research, offering insights into metabolism, nutritional status, and disease biomarkers. Rapid, reliable quantification of multiple amino acids in biological samples is critical for clinical studies, metabolomics, and quality control workflows.

Study Objectives and Overview

This application note demonstrates the capabilities of the research-only Kairos Amino Acid Kit for quantifying 20 physiologically relevant amino acids in solution. The method employs UPLC separation on a CORTECS UPLC C18 column and detection by an ACQUITY QDa mass detector in under 10 minutes. Performance was evaluated through solution-based panels and analysis of NIST SRM 2389a.

Methodology

Sample preparation involves protein precipitation in 0.1 M HCl, internal standard addition, and derivatization using AccQ-Tag Ultra 3X reagent and borate buffer. Chromatographic separation uses a 1.6 μm, 2.1 × 150 mm CORTECS UPLC C18 column at 55 °C with a water/acetonitrile gradient (0.1% formic acid). Detection is performed in positive electrospray mode, using single ion recording (SIR) for each amino acid and its 13C/15N-labeled internal standard. Calibration spans 5–4000 μM (or 2.5–500 μM for select analytes), with lower limits of quantification (LOQs) determined by an S/N >10 and precision ≤20% CV.

Used Instrumentation

• ACQUITY UPLC H-Class System
• CORTECS UPLC C18 1.6 μm, 2.1 × 150 mm Column
• ACQUITY QDa Mass Detector
• MassLynx v4.1 Software with TargetLynx Application Manager

Key Results and Discussion

No significant carryover was observed (<20% response in blanks). Within-day precision across replicate panels was ≤12% CV and between-day precision ≤15% CV, with mean CVs under 11.3%. Linearity was excellent (r2 >0.99) across the calibrated range, and back-calculated values were within ±15% (±20% at LLOQ). LOQs ranged from 1 μM to 4 μM with S/N ratios above 30. Accuracy tests yielded biases within ±12% for solution panels and ±8.9% for diluted NIST SRM 2389a. The method resolved critical isobaric pairs such as leucine/isoleucine and alanine.«

Benefits and Practical Applications

• Rapid run time under 10 minutes for high throughput
• Derivatization-based reverse-phase UPLC avoids ion-pair reagents
• Single kit for robust quantitation of 20 amino acids
• Stable derivatized samples compatible with general UPLC-MS systems
• Applicable in clinical research, metabolomics, and QA/QC labs

Future Trends and Opportunities

Expanding the kit to cover additional amino acids and low-abundance analytes could enhance its utility. Integration with high-resolution MS, automated sample prep, and data processing pipelines will facilitate larger metabolomic studies. Potential applications include biomarker discovery, nutritional monitoring, and precision medicine.

Conclusion

The Kairos Amino Acid Kit combined with ACQUITY UPLC and QDa detection provides a fast, accurate, and flexible platform for quantifying 20 amino acids in under 10 minutes. Its robust performance, minimal carryover, and simplified workflow make it well suited for a range of biomedical research applications.