Determination Of 18 Free Amino Acids In Serum And Drinks By High Performance Liquid Chromatography After Ultraviolet Derivatization With 2,4-Dinitrochlorobenzene

Purpose: To establish a method of determination of 18 free amino acids[aspartic acid(Asp), alutamic acid (Glu), histidine (His), serine(Ser), arginine(Arg), glycine(Gly), threonine(Thr), proline (Pro), alanine (Ala), valine(Val), methionine (Met), cystine (Cys), isoleucine (He), leucine (Leu), tryptophane (Trp), phenylalanine (Phe), lysine (Lys), tyrosine (Tyr)] in serum and drinks (wine, beer, grape juice and orange juice) by pre-column derivatization-high performance liquid chromatography. Method: After removing protein in serum with HPO₃ and removing precipitate in drink by centrifuging, the free amino acids in supernatant were derivatizated with 2,4-dinitrochlorobenzene in alkalescent media (pH9.0) in a water bath of 90℃. Then the derivatives were separated by C₁₈ column and detected by UV detector, and the amount of amino acids was quantified with internal or external standard method. Analytical column: phenomenex (USA), C₁₈(ODS) , 5μm, 250 × 4.6mm; column temperature:40℃; mobile phase A: acetonitrile, mobile phase B:HAc-NaAc (pH5.20); gradient elution with flow rate of 1.00ml/min; detection wavelength: 360nm.Results: The correlation coefficients of 18 free amino acids' regression equations detecting by internal or external standard method were 0.9951～ 0.9999; the range of RSD was 1.39%～4.67%; the detection limits of serum samples were: 0.20～7.72μg/ml,the detection limits of drink samples were 0.16～5.86μg/ml; the recoveries for serum samples were 65.9%～118% and for...