Determination of 2,6-diaminopimelic acid in bacteria, ruminal and duodenal digesta using HPLC with fulurescence or UV detection

摘要

A high-performance liquid chromatography method with pre-column derivatization was tested and used in the analysis of partially separated 2,6-diaminopimelic acid (DAPA) in rumen bacteria, duodenal digesta and feeds incubated in vitro with rumen fluid. The samples of analyzed materials were hydrolyzed with 6M HCL for 20 h at 104+-2 deg C. DAPA was determined after pre-column derivatization with o-phthaldialdehyde (OPA) in the presence ethanethiol (ESH). Separation of converted DAPA was caried out using a reversed-phase column (Nova-Pak C-18, 4 #mu#m, 250 x 4.6 mm I. D., Waters) by a binary gradint program and fluorescence or UV detection. The converted DAPA (as two peaks) was fluorescently monitored at an excitation wavelength of 229 nm, with 470 x nm cutoff-filter (the retention times: 33.83+-0.16 and 34. 43+-0.16 min), while the UV detector was set a 229 nm (retention times: 33.75+-0.16 and 34.36+-0.16 min). The DAPA paeks were completely resolved from interfering species in about 41 min. After 41 min. the average analytical recoveries were 98.4+-3.1% with fluorescence detection and 96.7+-4.0% with UV detection for total DAPA. The low within and between run coefficients of variations, high recoveries and low detection (2.05 nmol/ml) and quantification (6.78 nmol/ml) limits, point to satisfactory precision, reproducibility, accuracy and sensitivity of the proposed method. The use of fluorescence detection and the sum of DAPA peaks give the method high precision and accuracy. The presented method enabled partial separation of DAPA metabolism within the rumen microbial ecosystem.