Determination of urinary and blood amino acids using high-performance liquid chromatography system / Эрүүл мэндийн лаборатори

ABSTRACT

Background@#Screening programs for the detection of inherited disorders of amino acid metabolism is mandatory in most countries. Various laboratory methods are used for this purpose. We tested a high-performance liquid chromatography method for the separation of amino acids in blood and urine samples. @*Materials and Methods@#All reagents were of the HPLC grade purity,water used for t he analysis was deionized and reagents and samples were ultrafiltrated using a micropartition system.</br> The analysis was performed using the HPLC system with two pumps, a C18 column and a UV detector. All evaporations were done using a vacuum concentrator.</br> Amino acids were derivatized using a solution of ethanol, water, triethylamine and phenyl isothiocyanate. The amino acid derivatives were separated using a linear gradient with two solvents solvent A (sodium acetate acetonitrile) and solvent B (water acetonitrile). </br> Amino acid standards of 20, 50, 100, 200, 500, 750 and 1000 µM were prepared in 1 mM hydrochloric acid. </br> The EDTA blood as well as urine samples were spun at 1500 g for 15 min and then ultracentrifuged at 1500 g for 30 min.@*Results@#Experiments with various chromatographic conditions showed that factors which influenced the amino acids separation were the type of columns, mobile phase composition, flow-rate, gradient programs and timings.</br> After studying the effects of changes in individual parameters of chromatographic conditions, the following method parameters were chosen pre-column derivatization agent –PITC, separation column – C18, mobile phase –solvent A sodium acetate acetonitrile (982) and solvent B water acetonitrile (4060), gradient – linear, flow-rate – 1.2 mL/min. With this method 22 amino acids were separated within 35 minutes.@*Conclusion@#The developed method is simple and can be used by medical laboratories for the detection of inborn errors of amino acid metabolism.