Determination of 2,6-diaminopimelic acid in rumen bacteria by various high-performance liquid chromatography methods with pre-column derivatization

New high-performance liquid chromatography methods were used in the analysi s of total or partly separated stereoisomers of 2,6-diaminopimelic acid (DA PA) in rumen bacterial samples after hydrolysis with HCl. DAPA was determin ed after pre-column derivatization with o-phthaldialdehyde (OPA) in the pre sence of 2-mercaptoethanol (E(OH)SH) or ethanethiol (ESH). DAPA derivatives were analyzed using a reversed-phase column with UV detection at 340 nm. T wo binary gradient programs were used for analysis of DAPA derivatives. The derivatization procedures are based on DAPA conversion with OPA/E(OH)SH (m ethod 1) or with OPA/ESH (method 2). The retention times were 26.87+/-0.19 and 27.81+/-0.24 min for DAPA stereoisomers (method 1) and 37.67+/-0.33 min for total DAPA derivatives (method 2). The new procedure of method 2 enabl ed clear separation of DAPA stereoisomers as one peak, allowing better reli ability of this method. The total run time of the HPLC analyses was 55 min. The analytical recoveries of DAPA standards added to bacterial samples ran ged from 96.5 to 102.1% for both methods. The low inter- and intra-assay co efficient of variation, high recoveries and tow limits of detection, point to satisfactory precision, accuracy and sensitivity of the reported methods . The combination of the pre-concentration procedure and HPLC separation by method 1 or 2 provides an adequate tool for determination of relatively lo w levels of DAPA in biological samples.