Determination of urinary and blood amino acids using high-performance liquid chromatography system
- VernacularTitle:Шээс болон цусан дахь амины хүчлийн агууламжийг тодорхойлох өндөр үзүүлэлтэт шингэний хроматографийн арга
- Author:
Khishigbuyan D
1
;
Enkhjargal Ts
1
;
Gantuya P
1
;
Sodnomtseren B
1
Author Information
1. National Centre for Public Health
- Publication Type:Journal Article
- Keywords:
amino acids;
high-performance liquid chromatography;
blood;
urine;
phenylisocyanate (PITC);
derivatization
- From:
Health Laboratory
2017;7(2):16-20
- CountryMongolia
- Language:Mongolian
-
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.
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.
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).
Amino acid standards of 20, 50, 100, 200, 500, 750 and 1000 µM were prepared in 1 mM hydrochloric acid.
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.
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 (98:2) and solvent B: water : acetonitrile (40:60), 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.
- Full text:HL-2017-7(2)-16-20.pdf
