A Combined MRM and SIM Method for Direct Quantitative Determination of Amino Acids in Various Samples on LC/MS/MS

Importance of the Topic

Quantitative profiling of amino acids is essential across disciplines such as clinical research, food quality control and nutritional science. Methods that avoid laborious derivatization steps accelerate throughput, reduce reagents and lower operational costs while maintaining sensitivity and specificity.

Objectives and Study Overview

This work aims to establish a rapid, direct quantification method for twenty proteinogenic amino acids using combined multiple reaction monitoring (MRM) and selected ion monitoring (SIM) on a triple quadrupole LC/MS/MS platform. The performance of a novel Imtakt Intrada Amino Acid column is evaluated on various sample types, including human plasma, serum, urine, wine, beer, vinegar, sports water and amino acid drink.

Methodology and Instrumentation

Sample Preparation:

• Standards: Individual stocks in 0.1 N or 1.0 N HCl; mixed and serially diluted in water.
• Biological and beverage matrices: Protein precipitation with methanol/acetonitrile (1:1) at 1:3 or 1:4 ratio, vortex, centrifuge at 13 000 rpm, filter and inject.

Chromatography:

• Column: Intrada Amino Acid (100 × 3 mm, 3 µm).
• Mobile Phase A: ACN/THF/25 mM ammonium formate/0.3 % formic acid (9 : 75 : 16 : 0.3, v/v).
• Mobile Phase B: ACN/100 mM ammonium formate (20 : 80, v/v).
• Gradient: 0–3 min (0 % B)→9 min (17 % B)→16–18 min (100 % B)→18.5 min (0 % B).
• Flow Rate: 0.6 mL/min; Oven: 35 °C; Injection: 2 µL.

Mass Spectrometry:

• Instrument: Shimadzu UFLC with LCMS-8040 triple quadrupole.
• Ionization: ESI (+); Interface 400 °C; DL 300 °C.
• Gas: Nebulizing N₂ at 3 L/min; Drying N₂ at 15 L/min; CID gas Ar at 230 kPa.
• Acquisition: Simultaneous MRM and SIM transitions for each amino acid.

Main Results and Discussion

The combined MRM-SIM approach improved detection sensitivity for low-response analytes, notably glycine. In MRM mode glycine had an LOQ of 25 nmol/mL, whereas SIM achieved 5 nmol/mL. Other amino acids (Thr, Asp, Ala, Ser, Cys) also showed enhanced sensitivity in SIM. Calibration curves across 0.05–100 nmol/mL displayed correlation coefficients >0.99 for most compounds. Repeatability and accuracy met typical bioanalytical requirements.
Application to diverse samples demonstrated robust separation and quantitation without derivatization. In human plasma and serum, glutamic acid and alanine were most abundant. Urine profiles varied by individual but consistently showed high glutamine and low proline. In beverages, red wine and beer contained moderate levels of branched-chain amino acids; vinegar required SIM to detect cystine. Commercial sports water and amino acid drinks matched labeled compositions for key amino acids.

Benefits and Practical Applications

• Direct analysis without derivatization simplifies workflow and reduces reagents.
• Combined MRM-SIM ensures both high specificity and improved sensitivity for critical analytes.
• Wide applicability across biofluids and food/beverage matrices supports routine QA/QC and research.

Future Trends and Potential Applications

Further developments may include integration with stable isotope-labeled standards for absolute quantification, high-throughput automation, and coupling with high-resolution MS for expanded metabolomic coverage. The column chemistry could be extended to polar metabolite panels, and the workflow adapted for clinical diagnostic assays and real-time process monitoring in biomanufacturing.

Conclusion

The combined MRM and SIM LC/MS/MS method using the Intrada Amino Acid column provides a fast, robust and sensitive platform for quantifying twenty amino acids in various matrices. Eliminating derivatization enhances throughput and cost efficiency while maintaining analytical performance suitable for research and industrial applications.

References

1. Yazawa I, Tachikawa H. Development of a novel amino acids analysis column for LC-MS without derivatization. Imtakt Corp. 2014.
2. Matsumoto K, Watanabe J, Yazawa I. Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS. ASMS 2014, TP 510.