A HPLC method with UV detection for analysing of 2,6-diaminopimelic acid in rumen bacteria and intestinal digesta

Abstract

A HPLC method with UV detection for quantification of 2,6-diaminopimelic acid (DAPA) in rumen bacteria, duodenal and ileal digesta and faeces is described. Biological samples were hydrolyzed with 6 M HCl for 20 h at 104±2°C. DAPA was separated after pre-column derivatization with o-phthaldialdehyde (OPA) in the presence of ethanethiol (ESH). DAPA derivative was analyzed using a reversed-phase C18 column (3 μm, 250 x 2.1 mm, I.D.) by a gradient elution program and the UV and fluorescence detections. The converted DAPA (as two peaks) was UV monitored at 230.7 and 337 nm (the retention times: 46.77±0.20 and 47.25±0.21 min). The total run time, including 10 min of the column re-equilibration, was 60 min. The average recoveries of DAPA standards added to biological samples were satisfactory: 99.32±3.78 and 99.00±4.25% with UV detections at 230.7 and 337 nm, respectively. The presented HPLC method with the UV detections at 230.7 and 337 nm offers the low intra- and inter-assay coefficient variations (ca 0.5 and ca 1.1%), high recoveries (∼100%), so this procedure gives satisfactory precision, reproducibility and accuracy. The use of the UV detection at 337 nm offers lower limits of detection (ca 0.28 nmol/ml) and quantification (ca 0.91 nmol/ml) than the UV monitoring at 230.7 nm and the fluorescence detection (ca 1.1 and ca 3.6 nmol/ml, respectively). However, UV measurements at 230.7 nm produced strongest signals as compared with UV signals at 337 nm and the fluorescence detections. The presented HPLC method with UV at 230.7 nm enabled quantified of DAPA as a largest signal, so, this HPLC mode can be applied for the estimation of ruminal bacterial protein supply to ruminants and for monitoring of bacterial contamination of examined materials.