Optimization and validation of the Kairos amino acid Kit for plasma amino acid monitoring in inherited metabolic disorder patients

Abstract

Purpose

Genetic disorders affecting amino acid metabolism are a significant subset of inherited metabolic disorders (IMDs). Plasma amino acid (PAA) analysis is used for the diagnosis and monitoring of these disorders in order to avoid development of severe symptoms. However, PAA assays are often lengthy in analysis time (>2h/sample) and some methods lack specificity and sensitivity. This project offers a novel solution through the optimization and clinical validation of the Kairos Amino Acid Kit by Waters Corp.

Design and methods

Using liquid chromatography with tandem mass spectrometry and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization, amino acid quantification has been achieved for 45 amino acids in 15 min of chromatography time per sample. The method utilizes reversed-phase chromatography via a high-strength silica C18 column. The optimized column chemistry enables strong hydrophobic retention, resolving all isobaric amino acids for individual mass-spectrometer quantification. Method validation protocols include linearity, sensitivity, precision, and bias.

Results

Clinical validation has been performed, indicating high reproducibility and clinical applicability. Linearity results demonstrated 42/45 amino acids with R² > 0.975 over a linear range of at least 5–1000 µmol/L. Spiked plasma calibrators demonstrated high recovery with R² > 0.971. Twenty-day precision testing demonstrated total coefficient of variation < 15 % and sensitivity testing confirmed all analytes have limits of detection < 5 µmol/L, indicating high analytical sensitivity and precision. Method comparison to Waters’ MassTrak Kit demonstrates acceptable bias and supports a future study to update reference intervals.

Conclusion

The clinical validation of the Kairos Amino Acid Kit for plasma amino acid monitoring highlights the potential of this novel method to enhance amino acid quantification in clinical laboratories for IMD management.