Convenient method of threonine, methionine and their related amino compounds by high-performance liquid chromatography and its application to rumen fluid

A high-performance liquid chromatographic procedure for the quantitative de termination of cysteine (Cys), homocysteine (Hcys), methionine sulfoxide (M SO), methionine sulfone (MSO2), homoserine (Hser), glycine (Gly), threonine (Thr), 2-aminobutyric acid (2AB), methionine (Met), cystathionine (Cysta) and its application to rumen fluid are described. The samples containing Th r, Met and other related amino compounds were derivatized with 9-fluorenylm ethyl chloroformate. The separation of compounds was accomplished with a me thanol gradient in 25 mM sodium citrate buffer (obtaining pH 6.40 and 3.80 by addition of 25 mM citric acid). All derivatized compounds were separated on a Mightysil RP-18 GP (150x4.6 mm I.D., 5 mu m particle size) column. Al l analytes were detected at 265 nm with UV detection. The limits of detecti on (mu M) (S/N ratio, 3:1) and quantification (mu M) (S/N ratio, 10:1) of C ys, Hcys, MSO, MSO2, Hser, Gly, Thr, 2AB, Met and Cysta were 0.50 and 1.68; 1.76 and 5.85; 0.85 and 2.88; 0.92 and 3.09; 1.04 and 3.52; 0.76 and 2.52; 0.65 and 2.18; 0.39 and 1.36; 0.31 and 1.03; 0.17 and 0.58, respectively. The recoveries of all compounds in rumen fluid were 97.93-102.3% in the wit hin-day study and 94.52-98.69% on different day (6 days) studies. The avera ge contents (mu M) of Cys, Gly, Thr, 2AB, Met and Cysta were 1.72, 45.6, 20 .0, 4.3, 2.11 and 3.42 before morning feeding. The concentration of Thr, 2A B and Cysta in rumen fluid tended to increase with time after feeding where as Met showed the opposite tendency. (C) 2000 Elsevier Science B.V. All rig hts reserved.