Reverse-phase liquid chromatographic determination of lysine in complete feeds and premixes, using manual precolumn derivatization.
A sample portion is hydrolyzed with 6N HCl for 23 h and cooled, the pH is adjusted to 7.7 with NaOH, and the solution is diluted with pH 7.7 borate buffer. An aliquot of the sample extract is derivatized with 9-fluorenylmethyl chloroformate (9-FMC). Lysine is separated from other amino acids by isocratic reverse-phase liquid chromatography (LC) using fluorescence detection: 260 nm excitation and 313 nm emission. The mobile phase is acetonitrile-0.1M acetic acid (pH 4.2) buffer (53 + 47). Linearity is satisfactory over a range of 0.4-24 micrograms/mL. Results from 9 feed samples (1.1-2.7% lysine) analyzed by both the LC method and an amino acid analyzer were not significantly different statistically. Recovery of standard lysine, spiked just before derivatization on these same 9 samples (in duplicate), was 100.9% with a coefficient of variation (CV) of 2.4%. A study of within-day and between-day method precision resulted in CVs of 1.1 and 1.8%, respectively. The variation of results was negligible when the method was tested for ruggedness on 7 factors.
Cottingham
L S
LS
Purdue University, Department of Biochemistry, West Lafayette, IN 47907.
Smallidge
R L
RL
D000821:Animal Feed / Q000032:analysis*; D002853:Chromatography, Liquid; D005374:Filtration; D006868:Hydrolysis; D007202:Indicators and Reagents; D008239:Lysine / Q000032:analysis*
D007202:Indicators and Reagents; D008239:Lysine