Measurement of plasma amino acids by Ultraperformance® Liquid Chromatography.

BACKGROUND

Since the early 1960s, quantitative amino acid analysis (AAA) has traditionally been performed using ion-exchange chromatography with post-column ninhydrin derivatization. This established technology has many advantages, including relatively low cost of operation and ease of use. However, analysis times of 120+ min and high maintenance requirements make this technology unsuitable for the modern clinical laboratories with a requirement for rapid turnaround times. The work described here is a summary of our experiences with a new approach to full profile analysis of physiological amino acids.

METHODS

Amino acids were derivatized in batches with a proprietary reagent, AccQTag®, which reacts with primary and secondary amines. The derivatized amino acids were separated using Ultraperformance® Liquid Chromatography (UPLC). In a prospective method comparison, quantitative plasma amino acid data obtained from approximately 170 patient samples using both the UPLC method and a traditional ion-exchange chromatography amino acid analyzer were evaluated.

RESULTS

The data obtained from the two methods were found to agree well. Correlation coefficients for the most important amino acids seen in inborn errors of metabolism, such as phenylalanine, tyrosine and branched chain amino acids varied from 0.8658 to 0.9932 with minor slope biases. This approach also reduced the run time from 120 to 45 min per sample using a sample volume of 0.1 mL, compared to the 0.5 mL volume required for ion-exchange chromatography.

CONCLUSIONS

This new approach for the full profile analysis of physiological amino acids has been shown to be a viable substitute for current ion-exchange methodologies. It provides substantial benefits including significant reductions in runtime and necessary sample volume for the investigation and monitoring of patients with metabolic disorders and for nutritional management of a variety of patients.