Advances in Physiological Amino Acid Analysis

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Paula Hong, Kendon Graham, Tom Wheat and Diane M. Diehl
Waters Corporation
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Objective: Amino acids analysis (AAA) is used as a tool by clinicians to research various medical conditions and the treatment of those conditions. This analysis is complex both because the number of required amino acids is large and the samples can contain a large and unpredictable array of interferences. The existing AAA methods have relatively long run times, limited sensitivity, high cost per sample, and can be unreliable. To develop a new analytical solution, all of these concerns must be addressed within a framework that yields an accepted “correct” answer in a robust and reliable method. A complete system approach was chosen. The solution includes a well-characterized amino acid derivatization chemistry, a tested UltraPerfomance LC® method for analysis of the derivatized amino acids, amino acid standards, chromatographic columns, eluents and software for both data acquisition and customized reporting.


Methods: The amino acids are derivatized with 6-aminoquinolyl-hydroxysuccinimidyl

carbamate (AQC). Following the pre-column derivatization of the analytes, separation and detection are achieved with a reversed-phase UPLC® column and TUV detector, respectively. The analysis is complete within approximately 45 minutes and allows for the identification and quantification of 42 amino acids and related compounds. These samples are automatically analyzed with assured performance methods and reports are generated using pre-defined software templates.


Results: The quality and robustness of the analysis was studied to ensure accurate identification and quantification of all the amino acids screened. The amino acid standard includes the common acidics, neutrals and basic amino acids mixtures, freshly supplemented with glutamine and alloisoleucine. Norvaline is included as an internal standard. The amino acids are identified by retention time relative to a standard. Retention time precision for a standard is typically 0.2% RSD between runs. Quantification by peak area, with comparison to a standard, shows between run precision of typically 1.3% RSD. The limit of detection for the method is observed to be 50 femtomole on column; this corresponds to 0.5 μmol/L of each amino acid in the starting sample. Linearity from 100 fmoles to 50 pmoles/μL is shown with <20% deviation from calculated values for the lower limit of 100 femtomoles on column, or 1 μmol/L of amino acid in sample. The method has been tested with plasma and urine samples. The method is compatible with several deproteinization procedures, including sulfosalicyclic acid (SSA) and ultrafiltration. The precision studies described involve plasma samples deproteinized with SSA, and containing norvaline as an internal standard. A standard derivatization uses the equivalent of 10-20μL of plasma or urine and can be scaled down where required. For plasma samples, retention time precision was typically 0.2% RSD between runs. The area precision of the deproteinized samples was typically 2% RSD between sample.

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