Determination of free- and protein primary amino acids in biological materials by high-performance liquid chromatography and photodiode array detection

Abstract

Work from this laboratory resulted in improved high-performance liquid chromatography methods for quantification of free- and protein primary amino acids in biological materials. Biological samples were hydrolyzed with 6 M HCl for 20 h at 104±2°C. Primary amino acids, with the exception of tryptophan, were separated after pre-column derivatization with o-phthaldialdehyde (OPA) in the presence of ethanethiol (ESH). Derivatized amino acids were analyzed using a Nova Pak C18 column (4 μm, 250 x 4.6 mm I.D., Waters) by quaternary gradient system I. Detection was carried out simultaneously using UV monitoring at 337 nm and fluorescence detection (λex/λem = 336/425 nm). Derivatized amino acids were completely resolved from all interfering species in about 50 min. HPLC system I with two detection modes can also be used for quantification of free primary amino acids in ovine blood plasma. A trace amount of cysteine as its OPA/ESH derivative can be quickly quantified (∼ 3.5 min) using an isocratic HPLC system with UV detection at 274 nm. Separation and quantification of tryptophan in alkaline hydrolysates was achieved without derivatization using the same HPLC column by temate gradient elution system II with UV monitoring at 219 nm or fluorescence detection (λex/λem = 280/360 nm). The obtained average recoveries of assayed amino acids added to biological samples were near 100% when UV and fluorescence detection were applied. Generally, fluorescence detection in comparison with UV monitoring offers lower limits of detection (0.04-1.25 vs 0.05-12.7 μmol·l-1) and quantification (0.12-11.7 vs 0.18-42.0 μmol·l-1), however, the sensitivity of the UV detection mode is satisfactory for accurate and precise quantification of free- and protein amino acids in biological materials. The HPLC systems with UV detection assured better resolution of amino acid peaks in comparison with fluorescence detection. Satisfactory purity of analytical amino acid peaks (near 100%) and precision of HPLC systems with both detection modes renders these procedures suitable for routine analysis of amino acid concentrations in large numbers of biological samples. HPLC system I enabled quantification of 2,6-diaminopimelic acid, so the current chromatographic system can also be applied for the estimation of bacterial protein production in ruminants.