Chromatographic peak purity, investigation

Fig. 3 Investigation of chromatographic peak purity by diode array detection (A) spectra of drug substance and impurity (B) coelution of a mixture containing about 10% impurity (C) coelution of a mixture containing about 0.5% impurity. The spectra were obtained in the peak maximum, at about 5% and 50% of each side of the peak. The normalization was performed with respect to the first spectrum at the peak front (a matchfactor of 1000 means identical spectra) using commercial software.

Fig. 4 Investigation of chromatographic peak purity by means of LC-MS. The top and bottom trace show the UV chromatogram at 240 nm and the extracted ion chromatogram for a mass-to-charge ratio (m/z) 325. Insets A and B display (two of the) mass spectra taken at different retention time ranges of the UV-peak. Optimizing the chromatographic conditions resulted in a separation of the impurity which amounts to 0.5% area at 240 nm (data not shown).

Critical separations in chromatography should be investigated at an appropriate level. For critical separations, selectivity can be demonstrated by the resolution of the two components that elute closest to each other. Peak purity tests using diode array or mass spectrometric detectors may be useful to show that the analyte chromatographic peak is not attributable to more than one component. 

In this technique, an aliquot of the sample under investigation is first chromatographed. A second aliquot of sample is incubated with the enzyme under the appropriate conditions of pH and temperature. After a suitable time period, the incubated mixture is chromatographed. The disappearance of the substrate peak and/or appearance of product peaks confirms the identity and purity of the chromatographic peak. In cases where specific enzymes may not be available for a certain substrate, less specific enzymes, such as phosphatases, can be used for the identification of classes of compounds. In addition, it is possible to use coupled enzyme assays to drive a reaction to completion and thus characterize a peak in the chromatogram. 

The study of manoyl oxide derivatives i.e. 7 and 8 in, Fig (7), (i.e ent-hydroxy and en/-acetoxy-3(3-manoyl oxides) isolated from Cistus creticus, by GC-MS resulted in only one peak indicative of the purity of the products [33]. From the H-NMR data it is clear that the 13-epi isomer was present in both derivatives [58,139]. The chromatographic data of the compounds 7 and 8 were recently published [33,63]. Hence, investigations have proven that, apart from the 13-epi isomer, there are more isomers with varying intensities, which correspond to isomers that arise from the different configuration of C-8 chiral center [33]. This isomer showing a different configuration at C-8 has been isolated from the volatile leaf oil of Alaska (yellow) cedar and its structure has been confirmed using spectroscopic methods as well as chemical reactions [150],... 

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