Measurement of total and separate stereoisomers of diaminopimelic acid in rumen bacteria by high-performance liquid chromatography.
New high-performance liquid chromatographies were examined and applied for the analyses of the total (with one peak) and separate three stereoisomers of 2,6-diaminopimelic acid (DAP) in hydrolysed mixed rumen bacteria. The methods start with the reaction of DAP with 1-fluoro-2,4-dinitrophenylalanine amide for derivatisation. A mixture of 0.05 M triethylamine phosphate (pH 3.0) and acetonitrile (72:28, v/v) was used as an isocratic mobile phase for the total DAP determination (method 1), and a mixture of both solutions (78.5:21.5, v/v) for the determination of the separate three stereoisomers of DAP (method 2). The flow-rate was 1 ml/min; column, Merck LiChrospher 100 RP-18 (250 x 4 mm I.D.) of 5 microns particle size; column temperature, 40 degrees C; wavelength of detector, 325 nm. The retention times were 17.5 min for total DAP (method 1), and 39.4, 63.1 and 77.7 for meso-, LL- and DD-DAP, respectively (method 2). Lysine can also be determined by method 2 and the retention time was 122.8 min. The minimum detectable limit was 2.5 microM. The average analytical recoveries were 98.6% for total DAP and 99.1%, 100.4% and 100.2% for meso-, LL- and DD-DAP, respectively. The content of total DAP of the hydrolysed rumen bacteria collected from three goats fed lucerne cube and concentrate ranged from 25.55 to 27.36 mumol/g bacterial DM (method 1). The contents of the separate stereoisomers of DAP in the hydrolysed rumen bacteria from the three goats ranged from 19.64 to 22.06 and from 4.98 to 5.21 mumol/g bacterial DM for meso- and LL-DAP, respectively (method 2). DD-DAP was not detected.
el-Waziry
A M
AM
Laboratory of Animal Nutrition and Biochemistry, Miyazaki University, Japan.
Tomita
Y
Y
Ling
J R
JR
Onodera
R
R
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003960:Diaminopimelic Acid / Q000032:analysis* / Q000302:isolation & purification; D005260:Female; D006041:Goats; D006868:Hydrolysis; D012417:Rumen / Q000382:microbiology*; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism
D003960:Diaminopimelic Acid