Abstract
A method for the determination of diaminopimelic acid (DAPA) concentrations in feeds and rumen digesta by reversed-phase high-performance liquid chromatography using precolumn derivatization with o-phthaldialdehyde and fluorimetric detection was developed. Samples were oxidized and hydrolysed prior to analyses by HPLC. Hydrogen peroxide and formic acid were used for oxidation; hydrolyses were performed using 3 M hydrochloric acid under vacuum at 120°C for 17 h. Oxidation allowed more space for DAPA-OPA peak elution and hydrochloric acid hydrolysis reduced sample clean-up and extended the column life. Hydrolysates were diluted, adjusted to pH 7 and filtered. A Beckman Model 507 autosampler with a precolumn derivatization cassette was used for the derivatization process and fluorimetric detection was used to measure the OPA derivatives. Samples were prepared in order to have on-column DAPA concentrations in the range 10–100 pmol. The relative recovery of the standard solutions added to the feed samples ranged from 98.4 to 102.8%. The reproducibility of the method was evaluated by the analysis of eight alfalfa hay samples and eight alfalfa hay samples incubated in the rumen for 48 h and they yielded relative standard deviations of 2.04% and 2.02%, respectively.
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