Dereplication separation methods

In order to carry out dereplication procedures, a number of general characterization techniques are routinely employed to build information about the natural product that can be compared with other characterized compounds or sets of compounds. These techniques include chromatographic and other separation methods, chemical, spectroscopic, and biological approaches. Essentially, the process involves a series of questions at each stage of the extraction based on characteristics of the compoimd that allow it to be assigned to either a known compound, a general class of compounds, or an unknown structure. No single piece of data is... 

Another separation technique that has shown utility as a prefractionation method for dereplication is countercurrent chromatography (CCC) or centrifugal partition chromatography (CPC). This separation method is based on the... 

Using this approach for natural products dereplication, data are routinely obtained from 40 gg of crude extract. Performance examples include the identification of 16 analogs of teicoplanin and 12 analogs of phenelfamycin from separate samples. The summary of results obtained for phenelfamycin is shown in Table 6.4. The correlation of fraction, retention time, and molecular weight provides the essential information for rapid dereplication and identification. The time required to dereplicate natural product samples is about 1 week with this LC/MS-based method compared to several weeks by previous methods that involve traditional isolation steps. The use of this LC/MS-based methodology results in greater clarity and confident decisions for proceeding with the full structural study of an active component derived from a culture. 

Gas chromatography is a powerful technique to dereplicate low to moderate polarity natural products especially when it is coupled with ESI-MS. The reproducibility of ionization and fragmentation pattern from GC/MS make it one of the most efficient and reliable techniques in structure dereplication. There are different GC/MS libraries commercially available that contain mass spectra of hundred thousands of compounds. Unfortunately, approximately 80% of all known natural compounds are nonvolatile or thermally unstable and therefore incompatible with GC/MS methods. Due to the diversity of HPLC columns and the broad selections of solvent combinations and gradients, HPLC is capable of separating almost any kind of natural products. The development of HPLC detectors and... 

Currently most mycotoxins are still assayed using TLC, which permits effective compound separation and characterization. When the compounds to be detected are fluorescent, a fluorodensitometry reads the plates quantitatively and objectively. However, the use of RPLC has expanded rapidly in recent years. The diversity of detection systems (DAD, fluorescence, and MS) permits identification and accurate assay of a great variety of these compounds. Recently, a database for 474 mycotoxins and fungal metabolites for dereplication by standardized LC-UV-MS has been reported. Sample preparation techniques include extraction of mycotoxins using mostly chloroform, acetone, or methanol, purification of the extract with cleanup methods, and concentration of the extract. 

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