A Novel LC MS/MS Method for the Direct Analysis of Underivatized Amino Acids In Human Plasma

Significance of the Topic

Amino acids play a central role in human physiology as building blocks of proteins and intermediates in key metabolic pathways such as the urea and citric acid cycles. Profiling amino acid levels in plasma is crucial for diagnosing inborn errors of metabolism, monitoring nutritional status and guiding therapeutic interventions. A streamlined, accurate method for direct analysis of underivatized amino acids addresses limitations of traditional derivatization techniques, offering faster turnaround and improved separation.

Objectives and Study Overview

This study aimed to develop and validate a rapid LC-MS/MS method for simultaneous quantification of 45 underivatized amino acids in human plasma. The workflow was designed to minimize sample preparation, achieve high chromatographic resolution in a 13-minute cycle and deliver robust performance across a wide concentration range relevant for both low-abundance and high-abundance analytes.

Methodology and Instrumentation

Sample Preparation:

• Protein precipitation: 50 µL plasma mixed with sulfosalicylic acid, centrifuged, supernatant spiked with isotopic internal standards and diluted in mobile phase B.
• Calibration standards prepared in 1× PBS at 1–500 µmol/L to match matrix salinity.

Instrumentation:

• LC System: Waters Acquity UPLC with Raptor Polar X HILIC/ion-exchange column (100×2.1 mm, 2.7 µm) and guard cartridge.
• Mobile phases: A = 0.5% formic acid, 1 mM ammonium formate in water; B = 0.5% formic acid, 1 mM ammonium formate in 90:10 acetonitrile:water.
• Gradient: 96% B (0–2 min) to 30% B (10 min), re-equilibration to 96% B by 13 min; flow rate 0.3 mL/min; injection 5 µL; column at 35 °C.
• MS Detection: Waters Xevo TQ-S triple quadrupole, scheduled MRM in positive ESI mode.

Results and Discussion

Chromatographic Performance:

• Complete baseline separation of isobaric pairs (leucine/isoleucine/allo-isoleucine; alanine/sarcosine) within a 13-minute run.
• Retention time consistency ensured by matching salting conditions between standards and samples.

Linearity and Range:

• All 45 analytes exhibited linear response (r² > 0.990) over 1–500 µmol/L with 1/x-weighted quadratic regression.
• Samples for glutamine and alanine required dilution due to physiological concentrations exceeding 500 µmol/L.

Accuracy & Precision:

• Three quality control levels (low, medium, high) analyzed across three days (n=9) showed recoveries within nominal ranges and %RSD <20% for all analytes.
• Selection of 28 isotopic internal standards compensated effectively for matrix effects.

Benefits and Practical Applications

• High-throughput amino acid profiling supports clinical diagnostics, nutritional monitoring and metabolic research.
• Direct underivatized analysis reduces sample handling, chemical waste and potential artifacts from derivatization.
• Fast cycle time and robust performance enable large cohort studies and routine laboratory workflows.

Future Trends and Potential Applications

• Automation of sample preparation and integration with robotic platforms for increased throughput.
• Extension of the method to other biofluids (urine, cerebrospinal fluid) and post-translational modifications.
• Application of advanced data analytics and machine learning to identify metabolic signatures in disease and therapy response.

Conclusion

The presented LC-MS/MS workflow enables direct and simultaneous quantification of 45 amino acids in human plasma with a simple sample preparation and a 13-minute analytical cycle. This method delivers high-throughput, accurate and precise results, making it highly suitable for clinical, research and industrial applications in metabolic profiling.