A high-performance liquid chromatography method for the detection of diaminopimelic acid in urine.
We describe a quantitative assay for diaminopimelic acid (DAP) in urine. It involves (i) hydrolysis of urine samples, (ii) purification by several different liquid chromatography steps, and (iii) analysis by high-performance liquid chromatography on a reversed-phase C18 column. Tritiated-DAP, the internal standard, allows one to precisely follow the DAP-containing fractions and to determine the yield during purification. Sensitive and relatively accurate quantification of DAP, with a threshold of 50 fmol, is based on ion-pairing properties of eluants and ortho-phthaldialdehyde derivatization. The presence of DAP in relevant fractions was confirmed by combined gas chromatography and mass spectrometry. The DAP concentration in adult human urine pooled over 24 h ranges from 0.69 to 2.01 microM, a result in fair agreement with previously published values obtained by ninhydrin derivatization or gas chromatography.
Institut de Génétique et de Biologie Microbiennes, Rue César-Roux 19, CH-1005 Lausanne, Switzerland.
G
G
Borruat
C A
CA
Roten
L B
LB
Fay
D
D
Karamata
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003960:Diaminopimelic Acid / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D008297:Male; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D009764:o-Phthalaldehyde / Q000302:isolation & purification / Q000652:urine
D003960:Diaminopimelic Acid; D009764:o-Phthalaldehyde